Compositions and methods relating to a c-terminal peptide of troponin i with activity as a myofilament ca2+ desensitizer

ABSTRACT

Methods and compositions for treating a disorder of cardiac muscle and/or skeletal muscle in a subject according to aspects of the present disclosure. Treatment methods include administering a therapeutically effective dose of a C-terminal portion of troponin I capable of reducing sensitivity of cardiac muscle and/or skeletal muscle to Ca 2+  without decreasing maximum force production. Assays for identification of compounds capable of reducing sensitivity of cardiac muscle and/or skeletal muscle to Ca 2+  without decreasing maximum force production are further provided.

REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 62/883,958, filed Aug. 7, 2019, the entire content of which isincorporated herein by reference.

GRANT REFERENCE

This invention was made with government support under Grant Nos.HL-127691 and 138007, awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Heart failure is the most common end stage condition of cardiovasculardiseases. Diastolic heart failure, i.e., heart failure with preservedejection fraction, HFpEF, is a challenging clinical conditioncharacterized by the inefficient filling of the heart chambers duringdiastole resulting in reduced stroke volume based on the Frank-Starlingmechanism, see V. Melenovsky et al., Journal of the American College ofCardiology 49(2) (2007) 198-207; H. A. Shiels et al., Journal ofExperimental Biology 211(13) (2008) 2005-2013; and D. G. Allen et al.,Journal of Molecular and Cellular Cardiology 17(9) (1985) 821-840.

Muscle contraction is vital in animal mobility and heart function.Skeletal and cardiac muscles are striated muscles in which contractionis generated by the interaction of sarcomeric thick and thin filamentsin the crossbridge ATPase cycle. The thick filaments are mainly composedof the motor protein myosin, while the thin filaments are composed ofactin and the regulatory proteins tropomyosin and troponin. The troponincomplex contains three protein subunits: the calcium-binding subunit(troponin C, TnC), the inhibitory subunit (troponin I, TnI), and thetropomyosin-binding subunit (troponin T, TnT). The contraction ofmyocytes is initiated by the rise of cytosolic Ca²⁺ that binds TnC andinduces a series of allosteric changes in troponin and the thin filamentto allow myosin heads to bind actin, which activates myosin ATPase andcrossbridge cycling to generate power strokes. Subsequent decline ofcytosolic Ca²⁺ results in dissociation of Ca²⁺ from troponin to returnthe thin filament to the inhibitory state, detachment of myosin headsfrom the thin filament, and relaxation of the myocyte.

The primary function of troponin as a Ca²⁺-regulated brake in thesarcomere involves the key function of TnI that is responsible for theinhibition of myosin ATPase and muscle relaxation. Encoded by homologousgenes, three muscle fiber type-specific isoforms of TnI have evolved invertebrates. With the exception that cardiac TnI has a unique N-terminalextension, the structures of cardiac, fast and slow skeletal muscle TnIisoforms are highly conserved, see for example, J.-P. Jin et al.,Biochemistry 40(8) (2001) 2623-2631; and J.-J. Sheng et al., Gene 576(1)(2016) 385-394. The C-terminal end segment of TnI encoded by the lastexon is one of the most conserved structures in the three isoforms andacross vertebrate species, see J.-J. Sheng et al., Gene 576(1) (2016)385-394 and FIG. 1.

Mutations in the C-terminal end segment of cardiac TnI are associatedwith cardiomyopathies, the majority of which present clinically withdiastolic dysfunction (i.e., hypertrophic cardiomyopathy, HCM; andrestrictive cardiomyopathy, RCM), see for example, F. I. Gambarin etal., Heart 94(10) (2008) 1257; A. Doolan et al., Journal of Molecularand Cellular Cardiology 38(2) (2005) 387-393; Q.-W. Lu et al., Morimoto,Journal of geriatric cardiology 10(1) (2013) 91-101; M. S. Parvatiyar etal., Journal of Biomedicine and Biotechnology 2010 (2010) 1-9; R. H.Willott et al., Journal of Molecular and Cellular Cardiology 48(5)(2010) 882-892; and J.-J. Sheng et al., Frontiers in Physiology 5 (2014)165. An extensively studied RCM mutation, R192H, (see FIG. 1) has beenshown to cause severe diastolic dysfunction of the heart, as describedin J. Du et al., Archives of Biochemistry and Biophysics 456(2) (2006)143-150. The hearts of transgenic mice expressing C-terminal 19 aminoacid-deleted cardiac TnI also demonstrated severely impaired diastolicfunction as described in A. M. Murphy et al., Science 287(5452) (2000)488-491. This site is a Ca²⁺-regulated structural and functional domainof the troponin complex with a saturable binding to tropomyosin in lowCa²⁺ state, as described in Z. Zhang et al., The FEBS Journal 278(18)(2011) 3348-3359, indicating a role in the inhibitory activity of TnIduring muscle relaxation. This segment has also been implicated as amobile domain that is able to dock to the actin thin filament in aCa²⁺-dependent manner, see K. Murakami et al., Journal of MolecularBiology 352(1) (2005) 178-201.

The C-terminal end segment of TnI was not resolved in the staticcrystallographic structures of troponin complexes of both cardiacmuscle, see S. Takeda et al., Nature 424(6944) (2003) 35, and skeletalmuscle, see M. V. Vinogradova et al., Proceedings of the NationalAcademy of Sciences 102(14) (2005) 5038-5043, potentially due to itsallosteric nature. On the other hand, the C-terminal end segment of TnIforms a conserved epitope structure that is recognized by a monoclonalantibody (mAb) TnI-1, see FIG. 1 and see S. Akhter et al., FEBS Open Bio5 (2015) 64-75. Consistent with its binding to tropomyosin when residingin troponin complex, this epitope is an exposed structure for affinitychromatographic isolation, see Z. Zhang et al., Biochemistry 45(38)(2007) 11681-11694, or immunoprecipitation of the entire troponincomplex, see Z.-B. Yu et al., Journal of Biological Chemistry 276(19)(2001) 15753-15760. Further supporting the functional importance of thefolded structure of the C-terminal end segment of TnI, the single aminoacid substitution RCM mutation R192H abolishes the epitope recognized bymAb TnI-1, see Y. Li et al., Journal of Molecular and CellularCardiology 62 (2013) 227-236.

After decades of intensive research and numerous clinical trials,specific and effective treatments for heart failure remain to bedeveloped, see C. Satpathy et al., American Family Physician 73(5)(2006) 841-846; and J.-W. Ha et al., Journal of CardiovascularUltrasound 17(3) (2009) 86-95. Pharmacological therapy for diastolicdysfunction is currently multifactorial and involves addressingdiuresis, heart rate control, reducing myocardial hypertrophy andventricular relaxation, see C. Satpathy et al., American FamilyPhysician 73(5) (2006) 841-846; J.-W. Ha et al., Journal ofCardiovascular Ultrasound 17(3) (2009) 86-95; and F. Aziz et al.,Journal of Clinical Medicine Research 5(5) (2013) 327-334. Beta blockershave been commonly utilized in the treatment of heart failure byenhancing ventricular filling and lowering vascular resistance; however,beta blockers are notably negative inotropes and thereby have thepotential to weaken force production, see P. Arlock, B. et al.,Scandinavian Cardiovascular Journal 39(4) (2005) 250-254; and M. R.Bristow et al., Journal of Cardiac Failure 7(2) (2001) 8-12. On theother hand, positively inotropic drug such as digitalis and other Ca²⁺enhancers drastically increase myocardial energetic expenditure withvery limited long-term benefit, see S. Sasayama, Cardiovascular Drugsand Therapy, 10(6) (1997) 703-709.

There is a continuing need for compositions and methods for treatmentthat reduces muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction to ameliorate conditions such as disorders of skeletal muscleand/or cardiac muscle, particularly heart failure. There is a continuingneed for compositions and methods targeting specific steps of thecardiac muscle contraction and relaxation cycle to treat heart failure,particularly diastolic heart failure.

SUMMARY OF THE INVENTION

Methods of treating a disorder of cardiac muscle and/or skeletal musclein a subject according to aspects of the present disclosure whichinclude administering a therapeutically effective dose of a C-terminalportion of troponin I capable of reduction of cardiac muscle and/orskeletal muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction. According to aspects of the present disclosure, the subjectis a human being.

Methods of treating a disorder of cardiac muscle and/or skeletal musclein a subject according to aspects of the present disclosure whichinclude administering a therapeutically effective dose of a C-terminalportion of troponin I capable of reduction of cardiac muscle and/orskeletal muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction, wherein the C-terminal portion of troponin I is or includesa peptide selected from: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:32, and a variant of any thereof.

Methods of treating a disorder of cardiac muscle and/or skeletal musclein a subject according to aspects of the present disclosure whichinclude administering a therapeutically effective dose of a C-terminalportion of troponin I capable of reduction of cardiac muscle and/orskeletal muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction, wherein the C-terminal portion of troponin I is or includesa variant of a peptide selected from: SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:32, wherein thevariant includes one or more conservative amino acid substitutionscompared to the peptide selected from: SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:32, and wherein thevariant is capable of reduction of cardiac muscle and/or skeletal musclesensitivity to Ca²⁺ without decreasing maximum force production.

Methods of treating a disorder of cardiac muscle and/or skeletal musclein a subject according to aspects of the present disclosure whichinclude administering a therapeutically effective dose of a C-terminalportion of troponin I capable of reduction of cardiac muscle and/orskeletal muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction, wherein the C-terminal portion of troponin I is or includesa variant of a peptide selected from: SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:32, wherein thevariant includes one or more conservative amino acid substitutionscompared to the peptide selected from: SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:32, wherein thevariant has at least 70% identity, at least 80% identity, at least 90%identity, or at least 95% identity to the peptide selected from: SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6,SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQID NO:32, and wherein the variant is capable of reduction of cardiacmuscle and/or skeletal muscle sensitivity to Ca²⁺ without decreasingmaximum force production.

Methods of treating a disorder of cardiac muscle and/or skeletal musclein a subject according to aspects of the present disclosure whichinclude administering a therapeutically effective dose of a C-terminalportion of troponin I capable of reduction of cardiac muscle and/orskeletal muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction, wherein the C-terminal portion of troponin I is or includesa variant of a peptide of SEQ ID NO:32, wherein the variant has at least22 amino acids, wherein the variant includes one or more conservativeamino acid substitutions compared to SEQ ID NO:32, wherein the varianthas at least 70% identity, at least 80% identity, at least 90% identity,or at least 95% identity to SEQ ID NO:32, and wherein the variant iscapable of reduction of cardiac muscle and/or skeletal musclesensitivity to Ca²⁺ without decreasing maximum force production.

Methods of treating a disorder of cardiac muscle and/or skeletal musclein a subject according to aspects of the present disclosure whichinclude administering a therapeutically effective dose of a C-terminalportion of troponin I capable of reduction of cardiac muscle and/orskeletal muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction, wherein the C-terminal portion of troponin I is or includesa variant of a peptide of SEQ ID NO:1, wherein the variant has at least23 amino acids, wherein the variant comprises one or more conservativeamino acid substitutions compared to SEQ ID NO:1, wherein the varianthas at least 70% identity, at least 80% identity, at least 90% identity,or at least 95% identity to SEQ ID NO:1, and wherein the variant iscapable of reduction of cardiac muscle and/or skeletal musclesensitivity to Ca²⁺ without decreasing maximum force production.

Methods of treating a disorder of cardiac muscle and/or skeletal musclein a subject according to aspects of the present disclosure whichinclude administering a therapeutically effective dose of a C-terminalportion of troponin I capable of reduction of cardiac muscle and/orskeletal muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction, wherein the C-terminal portion of troponin I is or includesa variant of a peptide of SEQ ID NO:3, wherein the variant has at least27 amino acids, wherein the variant comprises one or more conservativeamino acid substitutions compared to SEQ ID NO:3, wherein the varianthas at least 70% identity, at least 80% identity, at least 90% identity,or at least 95% identity to SEQ ID NO:3, and wherein the variant iscapable of reduction of cardiac muscle and/or skeletal musclesensitivity to Ca²⁺ without decreasing maximum force production.

According to aspects of the present disclosure, one or more additionaltherapeutic agents is administered to the subject to treat a disorder ofcardiac muscle and/or skeletal muscle in the subject.

According to aspects of the present disclosure, administering atherapeutically effective dose of a C-terminal portion of troponin I,includes administering an expression cassette, wherein the expressioncassette includes a nucleic acid encoding the C-terminal portion oftroponin I, operably linked to a promoter. According to aspects of thepresent disclosure, the promoter is capable of driving expression of thenucleic acid encoding the C-terminal portion of troponin I in cardiacmuscle and/or skeletal muscle. According to aspects of the presentdisclosure, the promoter is a cardiac muscle protein promoter or askeletal muscle protein promoter. According to further aspects of thepresent disclosure, the promoter is selected from the group consistingof: human cardiac troponin I (hcTnI) promoter, human cardiac troponin T(hcTnT) promoter, human cardiac myosin-binding protein C promoter, humancardiac myosin light chain 2V promoter, human alpha cardiac myosin heavychain promoter, and human beta cardiac myosin heavy chain promoter.

According to aspects of methods of treating a disorder of cardiac musclein a subject according to aspects of the present disclosure, the subjecthas heart failure.

According to aspects of methods of treating a disorder of cardiac musclein a subject according to aspects of the present disclosure, the subjecthas a cardiac disorder relating to mutation of troponin or othersarcomeric proteins, such as hypertrophic cardiomyopathy (HCM),restrictive cardiomyopathy (RCM), and dilated cardiomyopathy (DCM).

According to aspects of methods of treating a disorder of skeletalmuscle in a subject according to aspects of the present disclosure, thesubject has a coordination disorder of the skeletal muscles.

Pharmaceutical compositions are provided according to aspects of thepresent disclosure which include a C-terminal portion of troponin Icapable of reduction of cardiac muscle and/or skeletal musclesensitivity to Ca²⁺ without decreasing maximum force production.

Pharmaceutical compositions are provided according to aspects of thepresent disclosure which include a nucleic acid encoding the C-terminalportion of troponin I capable of reduction of cardiac muscle and/orskeletal muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction.

Pharmaceutical compositions are provided according to aspects of thepresent disclosure which include a nucleic acid encoding the C-terminalportion of troponin I capable of reduction of cardiac muscle and/orskeletal muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction, wherein the nucleic acid is included in an expressioncassette and is operably linked to a promoter.

Pharmaceutical compositions are provided according to aspects of thepresent disclosure which include a nucleic acid encoding the C-terminalportion of troponin I capable of reduction of cardiac muscle and/orskeletal muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction, wherein the nucleic acid is included in an expressioncassette and is operably linked to a promoter, wherein the promoter iscapable of driving expression of the nucleic acid encoding theC-terminal portion of troponin I in cardiac muscle and/or skeletalmuscle.

Pharmaceutical compositions are provided according to aspects of thepresent disclosure which include a nucleic acid encoding the C-terminalportion of troponin I capable of reduction of cardiac muscle and/orskeletal muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction, wherein the nucleic acid is included in an expressioncassette and is operably linked to a promoter, wherein the promoter iscapable of driving expression of the nucleic acid encoding theC-terminal portion of troponin I in cardiac muscle and/or skeletalmuscle, and wherein the expression cassette is included in a vector.

Assays for identification of a test compound capable of reduction ofcardiac muscle and/or skeletal muscle sensitivity to Ca²⁺ withoutdecreasing maximum force production are provided according to aspects ofthe present disclosure which include contacting human alpha-tropomyosin,or a non-human homologue thereof, with a test compound under conditionsthat promote specific interaction between the tropomyosin and the testcompound, and detecting a change in reduction of cardiac muscle and/orskeletal muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction, if present.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a diagram showing the highly conserved C-terminal end segmentof TnI. Linear structures of the three isoforms of human TnI are shownschematically with their exon organizations, major functional sites andthe location of mAb TnI-1 epitope. The regions resolved in the crystalstructure of human cardiac troponin and chicken fast skeletal muscletroponin are shaded. The highly conserved amino acid sequence alignmentof the exon 8-encoded C-terminal end segment of cardiac (cTnI), slowskeletal muscle (ssTnI) and fast skeletal muscle (fsTnI) isoforms of TnIfrom representative vertebrate species demonstrates the conserved corestructure. The most conserved residues are bolded. The known mutationsites that cause hypertrophic cardiomyopathies (HCM), dilatedcardiomyopathies (DCM) and restrictive cardiomyopathies (RCM) arehighlighted in gray indicate changes in the conserved region would causeimpaired cardiac function (although they do not completely abolishtroponin function as non-lethal mutations including the most severemutation R192H shown in the bottom). Sequences shown in FIG. 1A include:Salmon cTnI—SEQ ID NO:4; Toad cTnI—SEQ ID NO:6; Chicken cTnI—SEQ IDNO:8; Mouse cTnI—SEQ ID NO:9; Bovine cTnI—SEQ ID NO:9; Human ssTnI—SEQID NO:10; Human fsTnI—SEQ ID NO:11; Human cTnI—aka HcTnT-C27—SEQ IDNO:3; Human Mutation Sites in SEQ ID NO:3 with highlighting to show HCMmutations; DCM Mutation Sites in SEQ ID NO:3 with highlighting to showDCM mutations; RCM Mutation Sites in SEQ ID NO:3 with highlighting toshow RCM mutations; and Human cTnI—R192H, also called HcTnT-C27-Hherein, in SEQ ID NO:3.

FIG. 1B is a diagram showing the crystal structure of cardiac troponincomplex is depicted (PDB ID: 1J1E) to illustrate the position of TnIC-terminal end segment (in the dashed oval) among the unresolvedregions.

FIG. 2A is a schematic diagram illustrating construction of plasmidsexpressing HcTnI-C27 and HcTnI-C27-H peptides. HcTnI-C27 and HcTnI-C27-Hpeptides were expressed as Tx3-SUMO-fusion proteins. The amino acidsequences of HcTnI-C27 and HcTnI-C27-H peptides, the Tx3-SUMO fusionprotein structure and the strategy of using AgeI cloning site at thefusion joint for the recovery of free peptide with zero fusion residueare illustrated. The N-terminal Tx3 tag in the fusion protein for metalaffinity purification is recognized by an mAb 3C11 for readyidentification. Sequences shown in FIG. 2A include CAGACCGGTGGAGAA (SEQID NO:24), a cloning vector sequence including an Agel restrictionendonuclease site; and GTCTGGCCACCTCTT (SEQ ID NO:25), a cloning vectorsequence complementary to SEQ ID NO:24, and including an Agelrestriction endonuclease site. Also shown is Human cTnI of SEQ ID NO:3with 4 amino acids at the N-terminus QTGGENREVGDWRKNIDALSGMEGRKKKFES,SEQ ID NO:26, to illustrate the SUMO protease cleavage site; andHcTnT-C27-H of SEQ ID NO:23 with 4 amino acids at the N-terminusQTGGENREVGDWHKNIDALSGMEGRKKKFES, SEQ ID NO:27, to illustrate the SUMOprotease cleavage site.

FIG. 2B is a schematic diagram illustrating construction of plasmidsexpressing HcTnI-C27 and HcTnI-C27-H peptides. The two-step PCR cloningprocedure to construct HcTnI-C27-H mutant cDNA into Tx3-SUMO vector isoutlined. Sequences shown in FIG. 2B include GACTGGCAACAAGAACATCGA, SEQID NO:28, a forward PCR primer specific for a nucleic acid encodingHuman cTnI R192H; and GAAGGAGGACACCGGTGGAGAAAACCGGGA, SEQ ID NO:29, aforward PCR primer specific for a nucleic acid encoding Human cTnI R192Hto introduce an Agel restriction endonuclease site into a PCR product asshown.

FIG. 3A is an image of an SDS-PAGE showing expression and purificationof Tx3-SUMO-HcTnI-C27 and Tx3-SUMO-HcTnI-C27-H fusion proteins andpeptide recovery. The SDS-gel shows an example of induced expression ofTx3-SUMO-HcTnI-C27 protein in E. coli and effective one-step Zn(II)column purification.

FIG. 3B is an image of an SDS-PAGE and two Western blots showingexpression and purification of Tx3-SUMO-HcTnI-C27 andTx3-SUMO-HcTnI-C27-H fusion proteins and peptide recovery. The Westernblots of purified fusion proteins showed that while mAb 3C11 bound toboth Tx3-SUMO-HcTnI-C27 and Tx3-SUMO-HcTnI-C27-H via the metal bindingtag in the fusion carrier, mAb TnI-1 has a strong binding toTx3-SUMO-HcTnI-C27 indicating preserved epitope structure, which isabolished in the Tx3-SUMO-HcTnI-C27-H mutant. MW, molecular weight.

FIG. 4A shows images of a Commassie Blue-stained SDS-PAGE, Amido Blackstained membranes after protein transfer from an SDS-PAGE, and Westernblots illustrating cleavage of C27 peptides from fusion proteins andpreservation of mAb TnI epitope in HcTnI-C27 peptide. The 15% small poreSDS-PAGE gels show the cleavage of Tx3-SUMO-HcTnI-C27 andTx3-SUMO-HcTnI-C27-H fusion proteins. Western blot using mAb 3C11against the metal-binding tag in carrier protein detected the intactfusion proteins and the cleaved carrier, as well as the recombinant SUMOprotease that also has the metal-binding tag for rapid purification.Western blot using mAb TnI-1 detected the HcTnI-C27 fusion protein andfree HcTnI-C27 peptide released by SUMO protease digestion but not thecarrier protein. The binding of mAb TnI-1 is lost for the HcTnI-C27-Hmutant peptide.

FIG. 4B shows images of an Amido Black stained membrane after proteintransfer from an SDS-PAGE and a Western blot illustrating cleavage ofC27 peptides from fusion proteins and preservation of mAb TnI epitope inHcTnI-C27 peptide. The preservation of mAb TnI-1 epitope in wild typebut not mutant C27 peptide was more clearly demonstrated by Western blotusing chemically synthesized peptides. Amido Black stains of the PVDFmembranes prior to blocking and mAb incubation are shown to verify theeffective blotting of the small peptides. MW, molecular weight.

FIG. 5A is a graph showing results of ELISA titration of mAb TnI-1against Tx3-SUMO-HcTnI-C27 and Tx3-SUMO-HcTnI-C27-H fusion proteins. Thefusion proteins were coated on microtiter plate to incubate with serialdilutions of the mAbs for ELISA titration as described in the methods.mAb TnI-1 recognizing the HcTnI-C27 epitope showed high affinity bindingto Tx3-SUMO-HcTnI-C27, which was significantly decreased but stillclearly detectable for Tx3-SUMO-HcTnI-C27-H. *P<0.0001 in Student'st-test.

FIG. 5B is a graph showing results of ELISA titration of mAb 3C11against Tx3-SUMO-HcTnI-C27 and Tx3-SUMO-HcTnI-C27-H fusion proteins. Thefusion proteins were coated on microtiter plate to incubate with serialdilutions of the mAbs for ELISA titration as described in the methods.mAb 3C11 titration curves against the metal tag of the fusion proteinsconfirmed comparable amounts of Tx3-SUMO-HcTnI-C27 andTx3-SUMO-HcTnI-C27-H coated on the microtiter plate.

FIGS. 6A and 6B are graphs showing similar affinities of mAb TnI-1 forHcTnI-C27 residing in SUMO fusion protein and in cardiac TnI. The ELISAtitration curves normalized to maximum binding in FIG. 6A showed thatmAb TnI-1 binds its epitope in Tx3-SUMO-HcTnI-C27 fusion protein and inwild type cardiac TnI (cTnI) with similar affinities as reflected by themAb TnI-1 dilutions for 50% maximum binding, shown in FIG. 6B, whichwere similarly decreased by the R to H single amino acid substitution inTx3-SUMO-HcTnI-C27-H fusion protein and in situ in R192H RCM mutantcardiac TnI; *P<0.005 compared with the wild type control in Student'st-test.

FIG. 7 is a graph showing isolated HcTnI-C27 peptide retains the bindingaffinity for tropomyosin. The competitive ELISA titration curvesnormalized to the maximum binding of intact cardiac TnI for tropomyosinwithout competition showed a dose-dependent competitive effect ofHcTnI-C27 peptide, *P <.05 in paired Student's t-test, which wasdiminished for the HcTnI-C27-H RCM mutant peptide.

FIG. 8A is a graph showing that synthetic and biologically madeHcTnI-C27 peptides have similar epitope conformation in physiologicbuffer. Normalized to the maximum binding of a predeterminedconcentration of mAb TnI-1 to intact cardiac TnI immobilized onmicrotiter plate, the competition curves of serial dilutions ofchemically synthesized and biologically made HcTnI-C27 peptides werenearly identical, reflecting their comparable epitope conformation in aphysiologic buffer.

FIG. 8B is a graph showing that synthetic and biologically madeHcTnI-C27-H mutant peptides both showed drastically diminished abilityin competing for mAb TnI-1, reflecting similarly altered conformation ofthe epitope.

FIG. 9 shows two graphs showing the Ca²⁺-desensitization effect ofHcTnI-C27 peptide in skinned mouse EDL muscle. Force-pCa curves ofskinned EDL muscle fibers at sarcomere length (SL) of 2.7 μm in theabsence or presence of 20 μM HcTnI-C27 peptide showed a decrease incooperativity (n) upon HcTnI-C27 treatment due to the decreases inmyofilament Ca²⁺ sensitivity in the activated state at higher Ca²⁺concentrations without change of maximum force production. Values arepresented as mean±SE. N=4 in each group. *P<0.05 vs the baseline inone-tail Student's t-test.

FIGS. 10A and 10B are graphs showing HcTnI-C27 peptide reduces Ca²⁺sensitivity in skinned rat cardiac muscle strips without decreasingmaximum force production. Ca²⁺-activated isometric force of skinned ratpapillary muscle at sarcomere length (SL) of 2.0 μm and 2.3 μm in theabsence or presence of 20 μM HcTnI-C27 peptide was plotted as Hillfitted force-pCa curves normalized to the maximum force at pCa 4.5. Atsarcomere length of 2.0 μm, HcTnI-C27 peptide resulted in a smallright-shift of the force-pCa curve (P=0.055 in one-tail Student t-test)as shown in FIG. 10A. As shown in FIG. 10B, at sarcomere length of 2.3μm, however, the addition of HcTnI-C27 peptide significantly decreasedCa²⁺ sensitivity and cooperativity (n), completely diminishing theCa²⁺-sensitization effect of increasing sarcomere length from 2.0 μm to2.3 μm. Values are presented as mean±SE. N=4 for SL 2.0 μm group and n=3for SL 2.3 μm group. The bar graphs show that the maximum forceproduction was not affected by the addition of HcTnI-C27 peptide.Statistical analysis was done using paired Student's t-test. pCa50, Ca²⁺concentration for 50% maximum force. *P<0.05 vs the SL 2.0 μm control;#P<0.05 vs the SL 2.3 μm baseline in the absence of HcTnI-C27 peptide; §P<0.05 vs SL 2.0 μm control in one-tail Student t-test.

FIGS. 11A and 11B are graphs showing that HcTnI-C27 reduces Ca²⁺sensitivity of skinned mouse cardiac muscle. The Force-pCa curves showthat 20 μM HcTnIC27 treatment decreased Ca²⁺-sensitivity of skinnedmouse ventricular papillary muscle at sarcomere lengths (SL) of 2.0 μm,FIG. 11A, and 2.3 μm, FIG. 11B, with decreased cooperativity (n) at SLof 2.0 μm. The maximum force production was not significantly affectedby the addition of HcTnIC27 peptide (the bar graphs). pCa50, Ca²⁺concentration for 50% maximum force. Values are presented as mean ±SE.N=3 for 2.0 μm and n=4 for 2.3 μm groups. Statistical analysis was doneusing paired Student's t-test. *P<0.05 vs the HcTnI-C27 peptide-absentbaseline.

FIG. 12 is a schematic image showing docking of HcTnI-C27 peptide ontropomyosin in molecular dynamic simulation. Representativeconformations of HcTnI-C27 peptide in two predicted binding locations oftropomyosin are shown with protein residues of tropomyosin labeled. Forclarity, the HcTnI-C27 peptide main chain is represented omitting the Oand H atoms. The two docking sites correspond to residues Cys190 and thesmall acidic patch Glu163-G1u164 in alpha-tropomyosin. The resultsdemonstrate a molecular conformation that favors the stabilization of ahelix-like conformation of tropomyosin based on interaction of anisolated C-terminal portion of troponin I, here shown as HcTnI-C27peptide, forming hydrophobic interactions driven by residues Trp191,Ile195 and Leu198 in HcTnI-C27.

DETAILED DESCRIPTION OF THE INVENTION

Scientific and technical terms used herein are intended to have themeanings commonly understood by those of ordinary skill in the art. Suchterms are found defined and used in context in various standardreferences illustratively including J. Sambrook and D. W. Russell,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress; 3rd Ed., 2001; F. M. Ausubel, Ed., Short Protocols in MolecularBiology, Current Protocols; 5th Ed., 2002; B. Alberts et al., MolecularBiology of the Cell, 4th Ed., Garland, 2002; D. L. Nelson and M. M. Cox,Lehninger Principles of Biochemistry, 4th Ed., W. H. Freeman & Company,2004; Herdewijn, P. (Ed.), Oligonucleotide Synthesis: Methods andApplications, Methods in Molecular Biology, Humana Press, 2004;Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins, 21st Ed., 2005; L. V. Allen, Jr. et al., Ansel's PharmaceuticalDosage Forms and Drug Delivery Systems, 8th Ed., Philadelphia, Pa.:Lippincott, Williams & Wilkins, 2004; and L. Brunton et al., Goodman &Gilman's The Pharmacological Basis of Therapeutics, McGraw-HillProfessional, 12th Ed., 2011.

The singular terms “a,” “an,” and “the” are not intended to be limitingand include plural referents unless explicitly stated otherwise or thecontext clearly indicates otherwise.

Described are compositions and methods of the present inventiongenerally relating to treatment of muscle disorders in a subject in needthereof. According to particular aspects, compositions and methods areprovided by the present invention relating to treatment of cardiacmuscle disorders in a subject in need thereof.

In specific embodiments, compositions and methods described hereinrelate to administration of an effective amount of a C-terminal portionof muscle troponin Ito a subject in need thereof. In specificembodiments, compositions and methods described herein relate toadministration of an effective amount of a C-terminal portion of muscletroponin I which includes the peptide of SEQ ID NO:1 or a variantthereof to a subject in need thereof. In specific embodiments,compositions and methods described herein relate to administration of aneffective amount of a nucleic acid encoding a C-terminal portion ofmuscle troponin I which includes the peptide of SEQ ID NO:1 or a variantthereof to a subject in need thereof.

The term “disorder of cardiac muscle” refers to conditions amelioratedby reduction of muscle sensitivity to Ca²⁺ without decreasing maximumforce production. Such conditions include, but are not limited to, heartfailure, cardiac disorders relating to mutation of troponin and othersarcomeric proteins, such as hypertrophic cardiomyopathy (HCM),restrictive cardiomyopathy (RCM), and dilated cardiomyopathy (DCM).

According to embodiments, disorders of skeletal muscle are treated byadministration of a C-terminal portion of muscle troponin I whichincludes the peptide of SEQ ID NO:1 or a variant thereof to a subject inneed thereof. Disorders of skeletal muscle treated by administration ofa C-terminal portion of muscle troponin I which includes the peptide ofSEQ ID NO:1 or a variant thereof include coordination disorders of theskeletal muscles.

In specific embodiments, compositions and methods described hereinrelate to administration of a C-terminal portion of troponin I whichincludes the peptide of SEQ ID NO:1 or a variant thereof and/or anucleic acid encoding the peptide or variant thereof to treat diastolicheart failure, i.e., heart failure with preserved ejection fraction,HFpEF.

A subject having a condition ameliorated by reduction of musclesensitivity to Ca²⁺ without decreasing maximum force production can beidentified by a medical professional using standard medical assessmenttechniques.

In specific embodiments, compositions and methods described hereinrelate to administration of a peptide including SEQ ID NO:1, or avariant of SEQ ID NO:1, the peptide including SEQ ID NO:1, or variantthereof, having a length of 23 amino acids to 200 amino acids,preferably having a length of 23 amino acids to 35 amino acids. Inspecific embodiments, compositions and methods described herein relateto administration of a nucleic acid encoding a peptide including SEQ IDNO:1, or a variant of SEQ ID NO:1, the peptide including SEQ ID NO:1, orvariant thereof, having a length of 23 amino acids to 200 amino acids,preferably having a length of 23 amino acids to 35 amino acids.

In specific embodiments, compositions and methods described hereinrelate to administration of a peptide including SEQ ID NO:2, or avariant of SEQ ID NO:2, the peptide or variant having a length of 23amino acids to 200 amino acids, preferably having a length of 23 aminoacids to 35 amino acids. In specific embodiments, compositions andmethods described herein relate to administration of a nucleic acidencoding a peptide including SEQ ID NO:2, or a variant of SEQ ID NO:2,and having a length of 23 amino acids to 200 amino acids, preferablyhaving a length of 23 amino acids to 35 amino acids.

In specific embodiments, compositions and methods described hereinrelate to administration of a peptide including SEQ ID NO:3, or avariant of SEQ ID NO:3, the peptide or variant having a length of 27amino acids to 200 amino acids, preferably having a length of 27 aminoacids to 35 amino acids. In specific embodiments, compositions andmethods described herein relate to administration of a nucleic acidencoding a peptide including SEQ ID NO:3, or a variant of SEQ ID NO:3,and having a length of 27 amino acids to 200 amino acids, preferablyhaving a length of 27 amino acids to 35 amino acids.

In specific embodiments, compositions and methods described hereinrelate to administration of a peptide including SEQ ID NO:4, or avariant of SEQ ID NO:4, the peptide or variant having a length of 27amino acids to 200 amino acids, preferably having a length of 27 aminoacids to 35 amino acids. In specific embodiments, compositions andmethods described herein relate to administration of a nucleic acidencoding a peptide including SEQ ID NO:4, or a variant of SEQ ID NO:4,and having a length of 27 amino acids to 200 amino acids, preferablyhaving a length of 27 amino acids to 35 amino acids.

In specific embodiments, compositions and methods described hereinrelate to administration of a peptide including SEQ ID NO:5, or avariant of SEQ ID NO:5, the peptide or variant having a length of 27amino acids to 200 amino acids, preferably having a length of 27 aminoacids to 35 amino acids. In specific embodiments, compositions andmethods described herein relate to administration of a nucleic acidencoding a peptide including SEQ ID NO:5, or a variant of SEQ ID NO:5,and having a length of 27 amino acids to 200 amino acids, preferablyhaving a length of 27 amino acids to 35 amino acids.

In specific embodiments, compositions and methods described hereinrelate to administration of a peptide including SEQ ID NO:6, or avariant of SEQ ID NO:6, the peptide or variant having a length of 33amino acids to 200 amino acids, preferably having a length of 33 aminoacids to 35 amino acids. In specific embodiments, compositions andmethods described herein relate to administration of a nucleic acidencoding a peptide including SEQ ID NO:6, or a variant of SEQ ID NO:6,and having a length of 33 amino acids to 200 amino acids, preferablyhaving a length of 33 amino acids to 35 amino acids.

In specific embodiments, compositions and methods described hereinrelate to administration of a peptide including SEQ ID NO:7, or avariant of SEQ ID NO:7, the peptide or variant having a length of 27amino acids to 200 amino acids, preferably having a length of 27 aminoacids to 35 amino acids. In specific embodiments, compositions andmethods described herein relate to administration of a nucleic acidencoding a peptide including SEQ ID NO:7, or a variant of SEQ ID NO:7,and having a length of 27 amino acids to 200 amino acids, preferablyhaving a length of 27 amino acids to 35 amino acids.

In specific embodiments, compositions and methods described hereinrelate to administration of a peptide including SEQ ID NO:8, or avariant of SEQ ID NO:8, the peptide or variant having a length of 33amino acids to 200 amino acids, preferably having a length of 33 aminoacids to 35 amino acids. In specific embodiments, compositions andmethods described herein relate to administration of a nucleic acidencoding a peptide including SEQ ID NO:8, or a variant of SEQ ID NO:8,and having a length of 33 amino acids to 200 amino acids, preferablyhaving a length of 33 amino acids to 35 amino acids.

In specific embodiments, compositions and methods described hereinrelate to administration of a peptide including SEQ ID NO:9, or avariant of SEQ ID NO:9, the peptide or variant having a length of 27amino acids to 200 amino acids, preferably having a length of 27 aminoacids to 35 amino acids. In specific embodiments, compositions andmethods described herein relate to administration of a nucleic acidencoding a peptide including SEQ ID NO:9, or a variant of SEQ ID NO:9,and having a length of 27 amino acids to 200 amino acids, preferablyhaving a length of 27 amino acids to 35 amino acids.

In specific embodiments, compositions and methods described hereinrelate to administration of a peptide including SEQ ID NO:10, or avariant of SEQ ID NO:10, the peptide or variant having a length of 27amino acids to 200 amino acids, preferably having a length of 27 aminoacids to 35 amino acids. In specific embodiments, compositions andmethods described herein relate to administration of a nucleic acidencoding a peptide including SEQ ID NO:10, or a variant of SEQ ID NO:10,and having a length of 27 amino acids to 200 amino acids, preferablyhaving a length of 27 amino acids to 35 amino acids.

In specific embodiments, compositions and methods described hereinrelate to administration of a peptide including SEQ ID NO:11, or avariant of SEQ ID NO:11, the peptide or variant having a length of 27amino acids to 200 amino acids, preferably having a length of 27 aminoacids to 35 amino acids. In specific embodiments, compositions andmethods described herein relate to administration of a nucleic acidencoding a peptide including SEQ ID NO:11, or a variant of SEQ ID NO:11,and having a length of 27 amino acids to 200 amino acids, preferablyhaving a length of 27 amino acids to 35 amino acids.

In specific embodiments, compositions and methods described hereinrelate to administration of a peptide including SEQ ID NO:32, or avariant of SEQ ID NO:32, the peptide or variant having a length of 22amino acids to 200 amino acids, preferably having a length of 22 aminoacids to 35 amino acids. In specific embodiments, compositions andmethods described herein relate to administration of a nucleic acidencoding a peptide including SEQ ID NO:32, or a variant of SEQ ID NO:32,and having a length of 22 amino acids to 200 amino acids, preferablyhaving a length of 22 amino acids to 35 amino acids.

The term “variant” refers to a peptide functional to reduce cardiacand/or skeletal muscle sensitivity to Ca²⁺ without decreasing maximumforce production of the cardiac and/or skeletal muscle and whichincludes an alteration, i.e. a substitution, insertion or deletion, ofone or more amino acids compared to the full-length amino acid sequenceof SEQ ID NO:1 while retaining a length in the range of 22 to 200 aminoacids, preferably having a length of 22 amino acids to 35 amino acids.The term “variant” refers to both naturally occurring variations of agiven peptide and recombinantly prepared mutations of a given peptide,wherein the variant is effective to reduce cardiac and/or skeletalmuscle sensitivity to Ca²⁺ without decreasing maximum force productionof the cardiac and/or skeletal muscle. Variants of SEQ ID NO:1, SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:32,have at least 80%, or at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater, amino acid sequenceidentity to a full length sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, or SEQ ID NO:32, wherein thevariant is effective to reduce cardiac and/or skeletal musclesensitivity to Ca²+without decreasing maximum force production of thecardiac and/or skeletal muscle according to aspects of the presentinvention.

Particular variants of the peptide of SEQ ID NO:1 are or include SEQ IDNO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7,SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:32.

(core - 23 amino acids) (SEQ ID NO: 1) EVGDWRKNIDALSGMEGRKKKFE(core variant - 23 amino acids) (SEQ ID NO: 2)(E/D)V(G/T)DWR(K/Q)N(I/V)(D/E)(A/E)(M/L/K) SGMEGRKK(K/M)F(E/D)Human cTnI - 27 amino acids. (SEQ ID NO: 3) ENREVGDWRKNIDALSGMEGRKKKFES(Salmon cTnI - 27 amino acids) (SEQ ID NO: 4)KKEEVTDWRQNVDAMSGMEGRKKMFDA (Toad cTnI - core 27 amino acids)(SEQ ID NO: 5) EIREVGDWRKNVDALSGMEGRKKKFES(Toad cTnI - core + C-terminal - 33 amino acids) (SEQ ID NO: 6)EIREVGDWRKNVDALSGMEGRKKKFESSGAVQT (Chicken cTnI - core 27 amino acids)(SEQ ID NO: 7) ESREVGDWRKNVDALSGMEGRKKKFEA(Chicken cTnI - core + C-terminal - 33 amino acids)  (SEQ ID NO: 8)ESREVGDWRKNVDALSGMEGRKKKFEAPGGGQG(mouse cTnI and bovine cTnI - 27 amino acids)  (SEQ ID NO: 9)ENREVGDWRKNIDALSGMEGRKKKFEG Human ssTnI - 34 amino acids.(SEQ ID NO: 10) RPVEVGDWRKNVEAMSGMEGRKKMFDAAKSPTSQHuman fsTnI - 29 amino acids. (SEQ ID NO: 11)DLRDVGDWRKNIEEKSGMEGRKKMFESES

As noted above, a peptide included in compositions and used in methodsaccording to aspects of the present invention has a length in the rangeof 22 to 200 amino acids, preferably having a length of 22 amino acidsto 35 amino acids. In addition to the core minimal peptide of 22 or 23amino acids or variants thereof detailed in SEQ ID NOs:1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, and 32, additional amino acids may be included at theC-terminal, N-terminal, or both the C-terminal and N-terminal of thecore peptide of SEQ ID NO:1 or variants according to aspects of thepresent invention and still retain the functional aspects of the coreminimal peptide or variants detailed in SEQ ID NOs:1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, and 32, specifically, efficacy to reduce cardiac and/orskeletal muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction of the cardiac and/or skeletal muscle. The additional aminoacids can be a particular sequence known to confer a specified function,such as a membrane transport sequence (MTS). Alternatively, theadditional amino acids can be portions of a C-terminal portion oftroponin I naturally contiguous with the core minimal peptide of 22 or23 amino acids or variants detailed in SEQ ID NOs:1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11 and 32.

According to aspects, a peptide for administration to a subject having acardiac and/or skeletal muscle disorder includes the sequences VGDWRKN(SEQ ID NO:30) and SGMEGRKK (SEQ ID NO:31). These sequences can belinked by a linker, such as a peptide linker or non-peptide linker.Thus, according to aspects, a peptide for administration to a subjecthaving a cardiac and/or skeletal muscle disorder includes the sequenceVGDWRKNXXXXSGMEGRKKKFE (SEQ ID NO:32) where each X is any amino acid.

Such additional amino acids can be added to a given amino acid sequenceby any of various methods including, but not limited to, recombinant DNAtechniques, chemical conjugation and photoconjugation techniques.

Peptides according to aspects of the present invention can be generatedby recombinant DNA techniques in vitro, ex vivo, or in vivo, accordingto aspects of the present invention.

As will be readily apparent to one of skill in the art, due to theredundancy of the genetic code, more than one nucleic acid encodes thepeptides according to aspects of the present invention, such as apeptide of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, or SEQ ID NO:32.

The term “nucleic acid” as used herein refers to RNA or DNA moleculeshaving more than one nucleotide in any form including single-stranded,double-stranded, oligonucleotide or polynucleotide. The terms“nucleotide sequence” is used to refer to the ordering of nucleotides inan oligonucleotide or polynucleotide in a single-stranded form ofnucleic acid.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotideshydrogen bonded to one another with thymine or uracil residues linked toadenine residues by two hydrogen bonds and cytosine and guanine residueslinked by three hydrogen bonds. In general, a nucleic acid includes anucleotide sequence described as having a “percent complementarity” to aspecified second nucleotide sequence. For example, a nucleotide sequencemay have 80%, 90%, or 100% complementarity to a specified secondnucleotide sequence, indicating that 8 of 10, 9 of 10 or 10 of 10nucleotides of a sequence are complementary to the specified secondnucleotide sequence. For instance, the nucleotide sequence 3′ -TCGA-5′is 100% complementary to the nucleotide sequence 5′-AGCT-3′. Further,the nucleotide sequence 3′-TCGA- is 100% complementary to a region ofthe nucleotide sequence 5′-TTAGCTGG-3′.

The terms “hybridization” and “hybridizes” refer to pairing and bindingof complementary nucleic acids. Hybridization occurs to varying extentsbetween two nucleic acids depending on factors such as the degree ofcomplementarity of the nucleic acids, the melting temperature, Tm, ofthe nucleic acids and the stringency of hybridization conditions, as iswell known in the art. The term “stringency of hybridization conditions”refers to conditions of temperature, ionic strength, and composition ofa hybridization medium with respect to particular common additives suchas formamide and Denhardt' s solution. Determination of particularhybridization conditions relating to a specified nucleic acid is routineand is well known in the art, for instance, as described in J. Sambrookand D. W. Russell, Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press; 3rd Ed., 2001; and F. M. Ausubel, Ed., ShortProtocols in Molecular Biology, Current Protocols; 5th Ed., 2002. Highstringency hybridization conditions are those which only allowhybridization of substantially complementary nucleic acids. Typically,nucleic acids having about 85-100% complementarity are considered highlycomplementary and hybridize under high stringency conditions.Intermediate stringency conditions are exemplified by conditions underwhich nucleic acids having intermediate complementarity, about 50-84%complementarity, as well as those having a high degree ofcomplementarity, hybridize. In contrast, low stringency hybridizationconditions are those in which nucleic acids having a low degree ofcomplementarity hybridize.

The terms “specific hybridization” and “specifically hybridizes” referto hybridization of a particular nucleic acid to a target nucleic acidwithout substantial hybridization to nucleic acids other than the targetnucleic acid in a sample.

Stringency of hybridization and washing conditions depends on severalfactors, including the Tm of the probe and target and ionic strength ofthe hybridization and wash conditions, as is well-known to the skilledartisan. Hybridization and conditions to achieve a desired hybridizationstringency are described, for example, in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001;and Ausubel, F. et al., (Eds.), Short Protocols in Molecular Biology,Wiley, 2002.

An example of high stringency hybridization conditions is hybridizationof nucleic acids over about 100 nucleotides in length in a solutioncontaining 6× SSC, 5× Denhardt's solution, 30% formamide, and 100micrograms/ml denatured salmon sperm at 37° C. overnight followed bywashing in a solution of 0.1× SSC and 0.1% SDS at 60° C. for 15 minutes.SSC is 0.15M NaCl/0.015M Na citrate. Denhardt's solution is 0.02% bovineserum albumin/0.02% FICOLL/0.02% polyvinylpyrrolidone.

Mutations can be introduced using standard molecular biology techniques,such as site-directed mutagenesis and PCR-mediated mutagenesis. One ofskill in the art will recognize that one or more amino acid mutationscan be introduced without altering the functional properties of thepeptide of SEQ ID NO:1, a peptide including SEQ ID NO:1, or a variant ofeither thereof. For example, one or more amino acid substitutions,additions, or deletions can be made without altering the functionalproperties of the peptide of SEQ ID NO:1, a peptide including SEQ IDNO:1, or a variant of either thereof, SEQ ID NO:2 or a variant thereof,SEQ ID NO:3 or a variant thereof, SEQ ID NO:4 or a variant thereof, SEQID NO:5 or a variant thereof, SEQ ID NO:6 or a variant thereof, SEQ IDNO:7 or a variant thereof, SEQ ID NO:8 or a variant thereof, SEQ ID NO:9or a variant thereof, SEQ ID NO:10 or a variant thereof, or SEQ ID NO:11or a variant thereof.

Conservative amino acid substitutions can be made in peptides having SEQID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 32 or including SEQ ID NO:1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 32 to produce variants according toaspects of the present invention. Conservative amino acid substitutionsare art recognized substitutions of one amino acid for another aminoacid having similar characteristics. For example, each amino acid may bedescribed as having one or more of the following characteristics:electropositive, electronegative, aliphatic, aromatic, polar,hydrophobic and hydrophilic. A conservative substitution is asubstitution of one amino acid having a specified structural orfunctional characteristic for another amino acid having the samecharacteristic. Acidic amino acids include aspartate, glutamate; basicamino acids include histidine, lysine, arginine; aliphatic amino acidsinclude isoleucine, leucine and valine; aromatic amino acids includephenylalanine, histidine, tyrosine and tryptophan; polar amino acidsinclude aspartate, glutamate, histidine, lysine, asparagine, glutamine,arginine, serine, threonine and tyrosine; and hydrophobic amino acidsinclude alanine, cysteine, phenylalanine, glycine, isoleucine, leucine,methionine, proline, valine and tryptophan; and conservativesubstitutions include substitution among amino acids within each group.Amino acids may also be described in terms of relative size, alanine,cysteine, aspartate, glycine, asparagine, proline, threonine, serine,valine, all typically considered to be small.

Variants according to aspects of the present invention can includesynthetic amino acid analogs, amino acid derivatives and/or non-standardamino acids, illustratively including, without limitation,alpha-aminobutyric acid, citrulline, canavanine, cyanoalanine,diaminobutyric acid, diaminopimelic acid, dihydroxy-phenylalanine,djenkolic acid, homoarginine, hydroxyproline, norleucine, norvaline,3-phosphoserine, homoserine, 5-hydroxytryptophan, 1-methylhistidine,3-methylhistidine, and ornithine.

To determine the percent identity of two amino acid sequences or of twonucleotide sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in the sequence of a first aminoacid or nucleic acid sequence for optimal alignment with a second aminoacid or nucleic acid sequence). The amino acid residues or nucleotidesat corresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position. Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences (i.e., % identity=numberof identical overlapping positions/total number of positions X100%). Inone embodiment, the two sequences are the same length. Alternatively,the two sequences may be different lengths, such as 1, 2, 3, 4, 5, 6, 7,8, 9 or 10 amino acids different in length. The additions or deletionsmay be at the N-terminus, C-terminus, internally or a mixture of anythereof.

The determination of percent identity between two sequences can also beaccomplished using a mathematical algorithm. A preferred, non-limitingexample of a mathematical algorithm utilized for the comparison of twosequences is the algorithm of Karlin and Altschul, 1990, PNAS 87:22642268, modified as in Karlin and Altschul, 1993, PNAS. 90:5873 5877. Suchan algorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searchesare performed with the NBLAST nucleotide program parameters set, e.g.,for score=100, wordlength=12 to obtain nucleotide sequences homologousto a nucleic acid molecules of the present invention. BLAST proteinsearches are performed with the XBLAST program parameters set, e.g., toscore 50, wordlength=3 to obtain amino acid sequences homologous to aprotein molecule of the present invention. To obtain gapped alignmentsfor comparison purposes, Gapped BLAST are utilized as described inAltschul et al., 1997, Nucleic Acids Res. 25:3389 3402. Alternatively,PSI BLAST is used to perform an iterated search which detects distantrelationships between molecules. When utilizing BLAST, Gapped BLAST, andPSI Blast programs, the default parameters of the respective programs(e.g., of XBLAST and NBLAST) are used. Another preferred, non-limitingexample of a mathematical algorithm utilized for the comparison ofsequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11 17.Such an algorithm is incorporated in the ALIGN program (version 2.0)which is part of the GCG sequence alignment software package. Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4is used.

The percent identity between two sequences is determined usingtechniques similar to those described above, with or without allowinggaps. In calculating percent identity, typically only exact matches arecounted.

Peptides and variants thereof according to aspects of the presentinvention can be produced in recombinant host cells using well-knownconventional techniques.

Broadly described, an expression cassette includes a nucleic acidencoding a peptide or variant thereof operably linked to one or moreregulatory elements that control transcriptional expression of thenucleic acid. An expression cassette can be introduced into a host cellwhere it is expressed. The expression cassette can be included in anexpression vector. The host cell can be in vitro, ex vivo, or in vivo.In the case where the host cell is in vitro or ex vivo, the peptide orvariant thereof can be isolated from the host cell and administered tothe subject in need thereof.

According to aspects of the present disclosure, the expression cassetteincludes a nucleic acid encoding a peptide including SEQ ID NO:1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 32, or a variant of any thereof.

According to aspects of the present disclosure, the expression cassetteincludes a nucleic acid encoding a peptide including SEQ ID NO:1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, or 32, wherein the nucleic acid includes SEQID NO:12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22.

For in vivo applications, the expression cassette, or expression vectorcontaining the expression cassette, is administered to the subject inneed thereof and expression of the peptide occurs in the subject. Thus,according to embodiments of the present invention, expression vectorincluding a nucleic acid encoding a peptide or variant thereof isadministered to a subject to treat disorders of skeletal muscle and/orcardiac muscle.

The term “regulatory element” as used herein refers to a nucleotidesequence which controls some aspect of the expression of an operablylinked nucleic acid. Exemplary regulatory elements illustrativelyinclude an enhancer, an internal ribosome entry site (IRES), an intron;an origin of replication, a polyadenylation signal (pA), a promoter, atranscription termination sequence, and an upstream regulatory domain,which contribute to the replication, transcription, post-transcriptionalprocessing of a nucleic acid. A secretory sequence encoding a secretionsignal that directs an encoded heterologous protein into the secretorypathway of a host cell is optionally included. Additional sequencesoptionally included in an expression vector include one or moresequences encoding a marker suitable for selection of cells carrying theexpression vector.

Those of ordinary skill in the art are capable of selecting and usingthese and other regulatory elements in an expression construct with nomore than routine experimentation.

The term “operably linked” as used herein refers to a nucleic acid infunctional relationship with a second nucleic acid. The term “operablylinked” encompasses functional connection of two or more nucleic acids,such as an oligonucleotide or polynucleotide to be transcribed and aregulatory element such as a promoter or an enhancer element, whichallows transcription of the nucleic acid to be transcribed.

The term “promoter” as used herein refers to a DNA sequence operablylinked to a nucleic acid to be transcribed such as a nucleic acidencoding a desired molecule. A promoter is generally positioned upstreamof a nucleic acid sequence to be transcribed and provides a site forspecific binding by RNA polymerase and other transcription factors.

Particular promoters included in operable linkage with a nucleic acidmolecule encoding a peptide or variant thereof are functional topreferentially express the operably-linked nucleic acid in a particularcell or tissue type. According to aspects of the present invention, anincluded promoter is a muscle-specific promoter, such as a skeletalmuscle-specific promoter or cardiac muscle-specific promoter. Accordingto aspects of the present invention, an included promoter is selectedfrom: human cardiac troponin I (hcTnI) promoter, human cardiac troponinT (hcTnT) promoter, human cardiac myosin-binding protein C promoter,human cardiac myosin light chain 2V promoter, human alpha cardiac myosinheavy chain promoter, and human beta cardiac myosin heavy chainpromoter.

Ubiquitous promoters can be used, such as viral promoters including, butnot limited to, CMV, SV40, and others described in detail in M. Y.Rincon, Cardiovascular Research, 108(1):4-20, 2015.

As will be recognized by the skilled artisan, the 5′ non-coding regionof a gene can be isolated and used in its entirety as a promoter in anexpression cassette to drive expression of an operably linked nucleicacid. Alternatively, a portion of the 5′ non-coding region can beisolated and inserted in an expression cassette to drive expression ofan operably linked nucleic acid. In general, about 500-6000 bp of the 5′non-coding region of a gene is included in an expression cassette toconfer expression of the operably linked nucleic acid. Assays which arewell-known in the art can be used to determine the ability of adesignated portion of the 5′ non-coding region of a gene to driveexpression of the operably linked nucleic acid.

Promoters described herein are known to be active in mammalian cardiacmuscle and/or skeletal muscle. Additional promoters useful in methodsand compositions of the present invention may be determined to be activein cardiac muscle and/or skeletal muscle of mammals and other speciesusing conventional techniques, such as analysis of expression of RNA orprotein produced from a nucleic acid expression construct in which thepromoter is operably linked to a nucleic acid encoding the RNA orprotein in cardiac muscle and/or skeletal muscle. Promoter homologuesand promoter variants can be included in an expression cassetteaccording to the present invention. The terms “promoter homologue” and“promoter variant” refer to a promoter which has substantially similarfunctional properties to confer the desired type of expression on anoperably linked nucleic acid compared to those disclosed herein.

The term “expression construct” is used herein to refer to adouble-stranded recombinant DNA molecule containing a nucleic aciddesired to be expressed and containing appropriate regulatory elementsnecessary or desirable for the transcription of the operably linkednucleic acid sequence in vitro or in vivo. The term “recombinant” isused to indicate a nucleic acid construct in which two or more nucleicacids are linked and which are not found linked in nature. The term“expressed” refers to transcription of a nucleic acid to produce acorresponding mRNA and/or translation of the mRNA to produce thecorresponding protein. Expression constructs can be generatedrecombinantly or synthetically or by DNA synthesis using well-knownmethodology.

An expression construct is introduced into a cell using well-knownmethodology, such as, but not limited to, by introduction of a vectorcontaining the expression construct into the cell. A “vector” is anucleic acid that transfers an inserted nucleic acid into and/or betweenhost cells becoming self-replicating. The term includes vectors thatfunction primarily for insertion of a nucleic acid into a cell,replication of vectors that function primarily for the replication ofnucleic acid, and expression vectors that function for transcriptionand/or translation of a nucleic acid. Also included are vectors thatprovide more than one of the above functions.

Expression vectors include plasmids, viruses, BACs, YACs, and the like.

Viral expression vectors can be used to express a desired protein orpeptide. Non-limiting examples of virus expression systems includeadenovirus, adeno-associated virus, herpes virus, vaccinia virus andlentivirus.

A host cell for expression of a peptide or variant thereof can beprokaryotic or eukaryotic, such as bacterial, plant, insect, fungus,yeast, and mammalian cells.

According to aspects of the present invention, the host cell is in vivo,such as in a human, so that the peptide or variant thereof is expressedin the host and treats a disorder of skeletal muscle and/or cardiacmuscle in the host.

An expression vector is introduced into a host cell using well-knowntechniques such as infection or transfection, including calciumphosphate transfection, liposome-mediated transfection, electroporation,sonoporation and nanoparticle-based methodologies. Expression constructsand methods for their generation and use to express a desired proteinare known in the art, as described, for example, in Sambrook et al.,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 2001; Ausubel, F. et al., (Eds.), Short Protocols in MolecularBiology, Wiley, 2002; and S. J. Higgins and B. D. Hames (Eds.), ProteinExpression: A Practical Approach, Oxford University Press, USA, 1999.

In addition to recombinant methodology, chemical synthetic techniquescan be used to produce a desired peptide or variant thereof. Forexample, a peptide or variant thereof can be produced using solid phasesynthesis, solution phase synthesis, partial solid phase synthesis orfragment condensation.

A membrane transport sequence (MTS) is optionally covalently ornon-covalently attached to a C-terminal portion of cardiac muscletroponin I capable of reduction of muscle sensitivity to Ca²⁺ withoutdecreasing maximum force production and/or a C-terminal portion ofskeletal muscle troponin I capable of reduction of muscle sensitivity toCa²⁺ without decreasing maximum force production of the presentinvention in order to facilitate transport of the peptide across cellmembranes and thereby facilitate intracellular delivery of the peptide.

MTS peptides which facilitate transport across cell membranes areexemplified by, and not limited to, Kaposi fibroblast factor, argininehomopolymer peptide, TAT peptides of HIV-1, Drosophila antennapediahomeodomain-derived peptide, herpes virus protein VP22 and transportanpeptide, see G. Guidotti et al., Cell-Penetrating Peptides: From BasicResearch to Clinics, 38(4): 406-424, 2017.

An MTS is coupled to a C-terminal portion of cardiac muscle troponin Icapable of reduction of muscle sensitivity to Ca²+without decreasingmaximum force production and/or a C-terminal portion of skeletal muscletroponin I capable of reduction of muscle sensitivity to Ca²⁺ withoutdecreasing maximum force production of the present invention by any ofvarious methods including, but not limited to, recombinant DNAtechniques, chemical conjugation and photoconjugation techniques.

The term “isolated” as used herein refers to a substance that has beenseparated from contaminating cellular components associated with thesubstance in nature not intended to be associated with the substance andthat would interfere with use of the substance in therapeutic,prophylactic, diagnostic or other uses. Generally, an isolated substancedescribed herein is at least about 80% pure, at least about 90% pure, atleast about 95% pure, or greater than about 99% pure. Purification isachieved using well-known standard methodology such as fractionationand/or chromatography, such as ammonium sulfate precipitation andelution chromatography such as size exclusion chromatography,displacement chromatography, ion exchange chromatography and bioaffinitychromatography. Exemplary purification methodology is described in S.Doonan, Protein Purification Protocols Humana Press, 1996.

In embodiments of the present invention, isolated peptide or variantthereof is administered to a subject and/or included in a composition ofthe present invention.

A C-terminal portion of troponin I included in methods andpharmaceutical compositions according to aspects of the presentinvention is “isolated” from the remaining portion of troponin I sincethe remaining portion of troponin I is not present in the pharmaceuticalcompositions and is not administered to a subject. A C-terminal portionof troponin I as described herein, isolated from the remaining portionof troponin I, is characterized by altered structure and functioncompared to a C-terminal portion of troponin I disposed in an intacttroponin I protein.

An isolated C-terminal portion of troponin I is characterized by alteredstructural dynamics which results in new functional characteristics,specifically, flexibility and capability to selectively desensitizeactivated myofilaments with increased relaxation and capability toreduce cardiac muscle and/or skeletal muscle sensitivity to Ca²+withoutdecreasing maximum force production.

According to aspects of the present disclosure, assays foridentification of a test compound capable of selectively desensitizingactivated myofilaments with increased relaxation are provided. Testcompounds identified as having activity to reduce cardiac muscle and/orskeletal muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction may be useful for treating a cardiac muscle disorder and/orskeletal muscle disorder as described herein.

Assays for identification of a test compound capable of selectivelydesensitizing activated myofilaments with increased relaxation areprovided according to aspects of the present disclosure which includecontacting human alpha-tropomyosin, or a non-human homologue thereof,with a test compound under conditions that promote specific interactionbetween the tropomyosin and the test compound, and detecting a change inreducing cardiac muscle and/or skeletal muscle sensitivity to Ca²⁺without decreasing maximum force production, if present.

The term “test compound” as used herein refers to an agent to bescreened for an ability to selectively reduce cardiac muscle and/orskeletal muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction. A test compound can be any of a variety of types ofcompounds, including chemical compounds, including small organic orinorganic molecules; biological macromolecules such as proteins,peptides, nucleic acids, oligonucleotides, polysaccharides,oligosaccharides; an extract of a biological material, including a cell,a tissue, a plant or plant part, an animal or animal part, bacteria,fungi, or other organisms or biological materials. A test compound whichis a small organic or inorganic molecule includes those having a size ofless than 500 Daltons, less than 750 Daltons, less than 1000 Daltons,less than 1250 Daltons, less than 1500 Daltons, less than 1750 Daltons,or less than 2000 Daltons.

Collections of test compounds may be used, such as small moleculelibraries or combinatorial libraries known in the art, and which may besynthesized using standard methodology or are commercially available.Test compounds in such collections may be assayed individually or asgroups of compounds. Optionally, the assay is configured as ahigh-throughput screening assay in which large numbers of test compoundsare assayed in parallel.

Test compounds may be based on modeling the 3-dimensional structure of aC-terminal portion of muscle troponin I and using rational drug designto provide compounds with similar molecular shape, size, charge andhydrophobicity characteristics.

A test compound can be included in an assay in any amount suitable fordetermining the effect of the compound in an assay for identification ofa test compound capable of selectively reducing cardiac muscle and/orskeletal muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction. The appropriate amount of the test compound will depend onvarious factors such as the particular assay type and the time periodover which the effect is assessed, and may be, without limitation, inthe range of about 0.001 nM to about 100 mM, about 0.01 nM to about 10mM, about 0.1 nM to about 1 mM, about 1 nM to about 100 uM, about 10 nMto about 10 uM, or about 100 nM to about 1 uM.

Assay methods according to aspects the present disclosure may be in vivocell-based assays, or in vitro non-cell-based biochemical or biophysicalassays, or in silico.

According to aspects of the present disclosure, assays foridentification of test compounds includes assay of the ability of a testcompound to selectively reducing cardiac muscle and/or skeletal musclesensitivity to Ca²⁺ without decreasing maximum force production ofmuscle cells. Such assays include administering a test compound to exvivo muscle, e.g. skinned muscle strips and/or skinned muscle sections,connected to a force transducer device and a length controller device,both of which devices can be obtained commercially, and measuring theeffect of the test compound on calcium-activated force and/orcontractility, for example at pCa 6.5, 6.3, 6.0, 5.8, 5.5, 5.0, and 4.5at 15° C.

According to aspects of the present disclosure, assays foridentification of test compounds includes one or more controls. Controlsare well-known in the art and one of skill in the art would readilyrecognize an appropriate control and be able to determine an appropriatecontrol for an assay method of the present disclosure with no more thanroutine experimentation. According to aspects of assay methods of thepresent disclosure, an effect of a test compound is compared to aneffect of an isolated C-terminal portion of muscle troponin I, such asan effect of an isolated C-terminal portion of muscle troponin I of SEQID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 32, or a variant of anythereof.

Test substances may be screened for ability to interact withalpha-tropomyosin or a portion containing the binding sites for theisolated C-terminal portion of muscle troponin I, in a cell e.g. in ayeast two-hybrid system.

As described and shown herein, a C-terminal portion of muscle troponin Iwhich is isolated from the remainder of muscle troponin I, e.g. bysynthesizing the C-terminal portion of muscle troponin I alone, ischaracterized by altered structural dynamics which results in newfunctional characteristics, specifically selectively desensitizedactivated myofilaments with increased relaxation. A specific newfunctional characteristic of the isolated C-terminal portion of muscletroponin I is the ability to “dock” with tropomyosin thereby reducingcardiac and/or skeletal muscle sensitivity to Ca²+without decreasingmaximum force production of the cardiac and/or skeletal muscle. As shownin the image representing molecular dynamic simulation of FIG. 12, theisolated C-terminal portion of muscle troponin I binds at two locationsof tropomyosin, the two docking sites correspond to residues Cys190 andthe small acidic patch Glu163-Glu164 in human alpha-tropomyo sin.

According to aspects of the present disclosure, assays foridentification of test compounds which specifically bind to the twodocking sites, i.e. binding sites, of alpha-tropomyosin, residue Cys190and residues Glu163-Glu164, in alpha-tropomyosin are provided accordingto aspects of the present disclosure which are useful to selectivelyreduce cardiac muscle and/or skeletal muscle sensitivity to Ca²+withoutdecreasing maximum force production. It is appreciated that analogousbinding sites in non-human alpha-tropomyosins will function similarlyand that these may be used in an assay according to aspects of thepresent disclosure.

The ability of a test compound to specifically bind to the two dockingsites of alpha-tropomyosin is assessed in vitro according to aspects ofthe present disclosure.

According to particular aspects of the present disclosure, an in vitroassay includes assay of in vitro motility of regulated actin filaments.Isolated and immobilized myosin motors are used to drive movement ofisolated F-actin-tropomyosin-troponin filaments under calcium regulationconditions. The effect of test compounds on a) velocity of the movement,and b) sensitivity of the actin filaments to calcium activation isassessed and may be compared to the effect of an isolated C-terminalportion of muscle troponin I, such as a peptide of SEQ ID NO:1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 32, ora variant of any thereof, on theseparameters.

According to particular aspects of the present disclosure, an in vitroassay includes a competition assay wherein a test compound is assayedfor the ability to displace an agent with known ability to bind tosegments including residue Cys190 and residues Glu163-Glu164 of humanalpha-tropomyosin, or analogous residues of a non-human homologuethereof, which binds isolated C-terminal portion of muscle troponin I,such as a peptide of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 32, ora variant of any thereof. According to particular aspects of the presentdisclosure, agent with known ability to bind to residue Cys190 andresidues Glu163-Glu164 of human alpha-tropomyosin, or analogous residuesof a non-human homologue thereof, is an antibody, or binding fragmentthereof.

According to particular aspects, an in vitro assay includes a detectionof signal modulation, wherein modulation of the signal is indicative ofspecific interaction of the test compound with the specific bindingsites for an isolated C-terminal portion of muscle troponin I in humanalpha-tropomyosin, or a non-human homologue thereof. According toparticular aspects, human alpha-tropomyosin, or non-human homologue, isdetectably labeled at one or both of the specific binding sites forC-terminal portion of muscle troponin I, i.e. residue Cys190 andresidues Glu163-Glu164. Specific binding of an isolated C-terminalportion of muscle troponin Ito the detectably labeled specific bindingsite(s) detectably decreases or increases the detected signal emanatingfrom the labeled tropomyosin. Assay of a test compound is performed bycontacting the test compound and detectably labeled humanalpha-tropomyosin, or non-human homologue, and then assaying for aspecific change in detectable signal due to the interaction of the testcompound with the labeled tropomyosin. The detectable label can be anylabel capable of attachment to a protein and which produces a detectablesignal modulated by specific binding at or near the site of attachmentof the label. According to particular aspects of the present disclosure,the tropomyosin is fluorescently labeled and the fluorescent signal isdetected by any of various fluorescence detection apparatus, such as aplate reader,fluorescence microscope, or a fluorometer.

The ability of a test compound to specifically bind to the two dockingsites of alpha-tropomyosin is assessed in silico according to aspects ofthe present disclosure.

A peptide functional to reduce cardiac and/or skeletal musclesensitivity to Ca²⁺ without decreasing maximum force production of thecardiac and/or skeletal muscle in a subject is administered to a subjectin need of treatment of a disorder of skeletal muscle and/or cardiacmuscle according to embodiments of the present invention. A peptidefunctional to reduce cardiac and/or skeletal muscle sensitivity to Ca²⁺without decreasing maximum force production of the cardiac and/orskeletal muscle in a subject is administered to a subject in need oftreatment of a disorder of skeletal muscle and/or cardiac muscleaccording to embodiments of the present invention, wherein the disorderis cardiac failure. A peptide functional to reduce cardiac and/orskeletal muscle sensitivity to Ca²⁺ without decreasing maximum forceproduction of the cardiac and/or skeletal muscle in a subject isadministered to a subject in need of treatment of heart failure,especially diastolic heart failure, i.e., heart failure with preservedejection fraction, HFpEF, according to embodiments of the presentinvention, wherein the disorder is cardiac failure.

The terms “treating” and “treatment” used to refer to reduction ofcardiac and/or skeletal muscle sensitivity to Ca²⁺ without decreasingmaximum force production of the cardiac and/or skeletal muscle in asubject.

A subject treated to reduce cardiac and/or skeletal muscle sensitivityto Ca²⁺ without decreasing maximum force production of the cardiacand/or skeletal muscle according to methods and using compositions ofthe present invention can be a mammalian subject. Humans are preferredsubjects treated according to methods and using compositions of thepresent invention. A mammalian subject treated according to aspects ofthe present invention can be any mammal including, but not limited to, anon-human primate; a rodent such as a mouse, rat, or guinea pig; adomesticated pet such as a cat or dog; a horse, cow, pig, sheep, goat,or rabbit. Birds can be treated according to aspects of the presentinvention, including poultry such as chickens, turkeys, and ducks.

In a particular embodiment of the present invention a method of treatinga muscle disorder by reducing cardiac and/or skeletal muscle sensitivityto Ca²⁺ without decreasing maximum force production of the cardiacand/or skeletal muscle in a subject is provided which includesadministering a therapeutically effective amount of a peptide or variantthereof to the subject.

A therapeutically effective amount is an amount which produces a desiredphysiologic or pharmacologic effect in a subject, specifically reducingcardiac and/or skeletal muscle sensitivity to Ca²⁺ without decreasingmaximum force production of the cardiac and/or skeletal muscle in asubject having a muscle disorder, such as heart failure.

Suitable dosages ranges of a peptide or variant thereof depend onvarious factors such as the age of the subject, the extent of muscledisorder in the subject, the general condition of the subject, the routeand form of administration of the composition being administered and theparticular composition administered. One of ordinary skill in the artwill be able to ascertain a therapeutically effective amount withoutundue experimentation in view of the present disclosure and what isknown in the art.

Administration of a peptide or variant thereof according to embodimentsof a method of the present invention includes administration accordingto a dosage regimen to produce a desired response. For example, one ormore dosage units of a peptide or variant thereof is administered to asubject at one time in particular embodiments. A suitable schedule foradministration of doses depends on several factors including age,weight, medical history and health status of the subject, type ofcomposition used and route of administration, for example. One of skillin the art is able to readily determine a dose and schedule ofadministration for a particular subject.

According to particular aspects of the present invention, anadministered amount of a peptide or variant thereof in the range ofabout 0.001 mg/kg body weight of the subject to about 100 mg/kg bodyweight of the subject, such as 0.001 mg/kg, 0.005 mg/kg, 0.01 mg/kg,0.05 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 5 mg/kg, 10 mg/kg, 50 mg/kg,or 100 mg/kg is administered and will have therapeutic efficacy. Theamount may be administered, for example, daily, twice daily, 3 timeseach day, 4 times each day, 6 times each day or more. The amount may beadministered, for example, daily, every other day, every three days,weekly, biweekly or monthly.

A peptide or variant thereof may be administered by any of variousroutes of administration, for example, oral, rectal, nasal, pulmonary,epidural, ocular, otic, intraarterial, intracardiac,intracerebroventricular, intracranial, intradermal, intravenous,intramuscular, intraperitoneal, intraosseous, intrathecal, intravesical,subcutaneous, topical, transdermal, and transmucosal, such as bysublingual, buccal, vaginal, and inhalational routes of administration.According to particular aspects of the present invention, a peptide orvariant thereof is administered intravenously.

Embodiments of the present invention optionally include administrationof a therapeutic agent in addition to a peptide or variant thereof.Non-limiting examples of pharmacologically active agents administered inaddition to a peptide or variant thereof in combination or separately,according to embodiments of methods of the present invention, includeantibiotics, antivirals, antineoplastic agents, analgesics,antipyretics, antidepressants, antipsychotics, anticancer agents,antihistamines, anti-osteoporosis agents, anti-osteonecrosis agents,antiinflammatory agents, anxiolytics, chemotherapeutic agents,diuretics, growth factors, hormones, non-steroidal anti-inflammatoryagents and vasoactive agents or a combination of any two or morethereof.

A pharmaceutically acceptable carrier can be included in a compositionincluding a peptide or variant thereof. A pharmaceutically acceptablecarrier is substantially non-toxic to the subject in amountsadministered and has substantially no deleterious effects on any activecomponent of a composition in which it is included.

The peptide or variant thereof to be administered is formulated fortopical, local and/or systemic administration to the subject.

Methods according to embodiments of the present invention includeadministration of a peptide or variant thereof as pharmaceuticalformulations, including those suitable for oral, rectal, nasal,pulmonary, epidural, ocular, otic, intraarterial, intracardiac,intracerebroventricular, intracranial, intradermal, intravenous,intramuscular, intraperitoneal, intraosseous, intrathecal, intravesical,subcutaneous, topical, transdermal, and transmucosal, such as bysublingual, buccal, vaginal, and inhalational routes of administration.

A pharmaceutical formulation of a peptide or variant thereof accordingto embodiments of the present invention is in any dosage form suitablefor administration to a subject, illustratively including solid,semi-solid and liquid dosage forms such as tablets, capsules, powders,granules, suppositories, pills, solutions, suspensions, ointments,lotions, creams, gels, pastes, sprays and aerosols.

Liposomes and emulsions are well-known types of pharmaceuticalformulations that can be used to deliver a pharmaceutical agent,particularly a hydrophobic pharmaceutical agent. In embodiments of thepresent invention, liposomes are particles typically produced as aunilammellar bilayer or a multilammellar bilayer of amphipathicmolecules enclosing an aqueous interior. Liposomes can include any typeof amphipathic materials compatible with a composition to be delivered,illustratively including naturally-occurring lipids, synthetic lipidsand combinations thereof.

A pharmaceutical formulation of a composition of the present inventioncan include a pharmaceutically acceptable carrier. The term“pharmaceutically acceptable carrier” refers to a carrier which issuitable for use in a subject without undue toxicity or irritation tothe subject and which is compatible with other ingredients included in apharmaceutical composition. Pharmaceutically acceptable carriers,methods for making pharmaceutical compositions and various dosage forms,as well as modes of administration are well-known in the art, forexample as detailed in Pharmaceutical Dosage Forms: Tablets, eds. H. A.Lieberman et al., New York: Marcel Dekker, Inc., 1989; and in L. V.Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, 8th Ed., Philadelphia, Pa.: Lippincott, Williams & Wilkins,2004; A. R. Gennaro, Remington: The Science and Practice of Pharmacy,Lippincott Williams & Wilkins, 21st ed., 2005, particularly chapter 89;and J. G. Hardman et al., Goodman & Gilman's The Pharmacological Basisof Therapeutics, McGraw-Hill Professional, 10th ed., 2001.

A solid dosage form for administration or for suspension in a liquidprior to administration illustratively includes capsules, tablets,powders, and granules. In such solid dosage forms, one or more activeagents, is admixed with at least one carrier illustratively including abuffer such as, for example, sodium citrate or an alkali metal phosphateillustratively including sodium phosphates, potassium phosphates andcalcium phosphates; a filler such as, for example, starch, lactose,sucrose, glucose, mannitol, and silicic acid; a binder such as, forexample, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose, and acacia; a humectant such as, forexample, glycerol; a disintegrating agent such as, for example,agar-agar, calcium carbonate, plant starches such as potato or tapiocastarch, alginic acid, certain complex silicates, and sodium carbonate; asolution retarder such as, for example, paraffin; an absorptionaccelerator such as, for example, a quaternary ammonium compound; awetting agent such as, for example, acetyl alcohol, glycerolmonostearate, and a glycol; an adsorbent such as, for example, kaolinand bentonite; a lubricant such as, for example, talc, calcium stearate,magnesium stearate, a solid polyethylene glycol or sodium laurylsulfate; a preservative such as an antibacterial agent and an antifungalagent, including for example, sorbic acid, gentamycin and phenol; and astabilizer such as, for example, sucrose, EDTA, EGTA, and anantioxidant.

Solid dosage forms optionally include a coating such as an entericcoating. The enteric coating is typically a polymeric material.Preferred enteric coating materials have the characteristics of beingbioerodible, gradually hydrolyzable and/or gradually water-solublepolymers. The amount of coating material applied to a solid dosagegenerally dictates the time interval between ingestion and drug release.A coating is applied having a thickness such that the entire coatingdoes not dissolve in the gastrointestinal fluids at pH below 3associated with stomach acids, yet dissolves above pH 3 in the smallintestine environment. It is expected that any anionic polymerexhibiting a pH-dependent solubility profile is readily used as anenteric coating in the practice of the present invention to achievedelivery of the active agent to the lower gastrointestinal tract. Theselection of the specific enteric coating material depends on propertiessuch as resistance to disintegration in the stomach; impermeability togastric fluids and active agent diffusion while in the stomach; abilityto dissipate at the target intestine site; physical and chemicalstability during storage; non-toxicity; and ease of application.

Suitable enteric coating materials illustratively include cellulosicpolymers such as hydroxypropyl cellulose, hydroxyethyl cellulose,hydroxypropyl methyl cellulose, methyl cellulose, ethyl cellulose,cellulose acetate, cellulose acetate phthalate, cellulose acetatetrimellitate, hydroxypropylmethyl cellulose phthalate,hydroxypropylmethyl cellulose succinate and carboxymethylcellulosesodium; acrylic acid polymers and copolymers, preferably formed fromacrylic acid, methacrylic acid, methyl acrylate, ammoniummethylacrylate, ethyl acrylate, methyl methacrylate and/or ethyl; vinylpolymers and copolymers such as polyvinyl pyrrolidone, polyvinylacetate, polyvinylacetate phthalate, vinylacetate crotonic acidcopolymer, and ethylene-vinyl acetate copolymers; shellac; andcombinations thereof. A particular enteric coating material includesacrylic acid polymers and copolymers described for example U.S. Pat. No.6,136,345.

The enteric coating optionally contains a plasticizer to prevent theformation of pores and cracks that allow the penetration of the gastricfluids into the solid dosage form. Suitable plasticizers illustrativelyinclude: triethyl citrate (Citroflex 2), triacetin (glyceryltriacetate), acetyl triethyl citrate (Citroflec A2), Carbowax 400(polyethylene glycol 400), diethyl phthalate, tributyl citrate,acetylated monoglycerides, glycerol, fatty acid esters, propyleneglycol, and dibutyl phthalate. In particular, a coating composed of ananionic carboxylic acrylic polymer typically contains approximately 10%to 25% by weight of a plasticizer, particularly dibutyl phthalate,polyethylene glycol, triethyl citrate and triacetin. The coating canalso contain other coating excipients such as detackifiers, antifoamingagents, lubricants (e.g., magnesium stearate), and stabilizers (e.g.hydroxypropylcellulose, acids or bases) to solubilize or disperse thecoating material, and to improve coating performance and the coatedproduct.

Liquid dosage forms for oral administration include one or more activeagents and a pharmaceutically acceptable carrier formulated as anemulsion, solution, suspension, syrup, or elixir. A liquid dosage formof a composition of the present invention may include a colorant, astabilizer, a wetting agent, an emulsifying agent, a suspending agent, asweetener, a flavoring, or a perfuming agent.

For example, a composition for parenteral administration may beformulated as an injectable liquid. Examples of suitable aqueous andnonaqueous carriers include water, ethanol, polyols such as propyleneglycol, polyethylene glycol, glycerol, and the like, suitable mixturesthereof; vegetable oils such as olive oil; and injectable organic esterssuch as ethyloleate. Proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of a desirableparticle size in the case of dispersions, and/or by the use of asurfactant, such as sodium lauryl sulfate. A stabilizer is optionallyincluded such as, for example, sucrose, EDTA, EGTA, and an antioxidant.

For topical administration, a composition can be formulated foradministration to the skin such as for local effect, and/or as a “patch”formulation for transdermal delivery. Pharmaceutical formulationssuitable for topical administration include, for example, ointments,lotions, creams, gels, pastes, sprays and powders. Ointments, lotions,creams, gels and pastes can include, in addition to one or more activeagents, a base such as an absorption base, water-removable base,water-soluble base or oleaginous base and excipients such as athickening agent, a gelling agent, a colorant, a stabilizer, anemulsifying agent, a suspending agent, a sweetener, a flavoring, or aperfuming agent.

Transdermal formulations can include percutaneous absorption enhancerssuch as acetone, dimethyl acetamide, dimethyl formamide, dimethylsulfoxide, ethanol, oleic acid, polyethylene glycol, propylene glycoland sodium lauryl sulfate. lonotophoresis and/or sonophoresis can beused to enhance transdermal delivery.

Powders and sprays for topical administration of one or more activeagents can include excipients such as talc, lactose and one or moresilicic acids. Sprays can include a pharmaceutical propellant such as afluorinated hydrocarbon propellant, carbon dioxide, or a suitable gas.Alternatively, a spray can be delivered from a pump-style spray devicewhich does not require a propellant. A spray device delivers a metereddose of a composition contained therein, for example, using a valve forregulation of a delivered amount.

Embodiments of inventive compositions and methods are illustrated in thefollowing examples. These examples are provided for illustrativepurposes and are not considered limitations on the scope of inventivecompositions and methods.

EXAMPLES

Materials and Methods

Cloning and Bacterial Expression of HcTnI-C27 and R192H Mutant

A fusion protein approach was employed to generate biologicallysynthesized cTnI-C27 peptide. cDNA encoding the C-terminal 27 aminoacids of human cardiac TnI (HcTnI-C27) was amplified by polymerase chainreaction (PCR) from a full length cDNA. The forward PCR primer containeda restriction enzyme Agel site followed by a Gly codon (GGA) requiredfor small ubiquitin-like modifier (SUMO) protease cleavage that leaveszero residue behind, see M.P. Malakhov et al., Journal of Structural andFunctional Genomics 5(1-2) (2004) 75-86; A.-M. Catanzariti et al.,Protein Science 13(5) (2004) 1331-1339; and C. Amarasinghe et al.,Protein and Peptide Letters 22(10) (2015) 885-892 and FIG. 2A. Thereverse PCR primer contained a BamHI restriction site previously used toclone the intact cDNA. The PCR product was double-digested with Agel andBamHI and purified using agarose gel electrophoresis for insertion intoa T7 RNA polymerase-based expression plasmid constructed with atransition metal-binding tag (Tx3) and a SUMO substrate domain, see C.Amarasinghe et al., Protein and Peptide Letters 22(10) (2015) 885-892,followed by an in-frame AgeI site and downstream multi-cloning sites.The DNA ligation product was used for transformation of JM109 competentE. coli cells and antibiotic resistant colonies were screened using PCR.Recombinant expression plasmids were miniprepped and sequence confirmed.

A recombinant plasmid expressing SUMO-fused HcTnI-C27 containing the RCMmutation R192H (HcTnI-C27-H) was constructed using PCR. A megaprimer wasfirst made by PCR from wild type human cardiac TnI cDNA template using aforward primer containing the mutant site paired with the same reverseprimer used above. This PCR-produced megaprimer was purified for use asthe reverse primer to pair with the forward primer containing the Agelrestriction site in a second PCR on wild type cDNA template (FIG. 2B).The final PCR product was double-digested using AgeI and BamHI, purifiedon agarose gel and cloned into Tx3-SUMO plasmid as above.

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE was performed to monitor the expression and identify the C27peptide in intact TnI and fusion protein constructs. Briefly, 14%Laemmli SDS-PAGE with an acrylamide:bisacrylamide ratio of 29:1 was usedto monitor protein expression. Protein samples were prepared bydissolving the proteins in an SDS-PAGE gel sample buffer containing 2%SDS and 3% (3-mercaptoethanol. The samples were run on 0.75 mm gels at20 mA per gel using a Bio-Rad mini-gel system. Resolved gels werestained with Coomassie Blue R-250 and de-stained using 10% acetic acid.The results were documented by imaging using a high resolution scanner.

Tris-Tricine small pore SDS-gels were utilized to resolve small.Briefly, 15% SDS-PAGE with acrylamide:bisacrylamide ratio of 20:1 wasused to monitor the recovery of peptides. Samples were prepared asabove. The gel was run using different anode and cathode buffers asdescribed in H. Schagger et al., 166(2) (1987) 368-379; J.-P. Jin,Journal of Biological Chemistry 270(12) (1995) 6908-6916. The runningconditions and documentation of results were carried out as above.

Western Blotting

The protein bands resolved on SDS-PAGE gels were electricallytransferred to nitrocellulose (NC) membranes or polyvinylidenedifluoride (PVDF) membranes using a Bio-Rad semidry electrotransferapparatus at 5 mA per cm². The membranes were then blocked with 1%bovine serum albumin (BSA) in Tris-buffered saline (TBS) at roomtemperature for 30 minutes, and incubated with mAb TnI-1 against anepitope in the C-terminal end of TnI or mAb 3C11 against the Tx3 metalaffinity purification tag at 4° C. overnight. The TnI-1 mAb was raisedusing standard hybridoma technology in which the immunization of mouseused purified intact chicken fast TnI. Detailed characterization asreported in J.-P. Jin et al., Biochemistry 40(8) (2001) 2623-2631demonstrated its specificity against the C-terminal end segment of TnI.The 3C11 mAb was generated using standard hybridoma technology in whichthe immunization used a Tx3 metal binding tag fusion protein. Detailedcharacterization as reported in R. Liu, Calponin and CytoskeletonDynamics in Macrophage Functions and the Pathogenesis ofAtherosclerosis, Wayne State University, Wayne State UniversityDissertations, 2016, demonstrated its specificity and high affinityagainst the Tx3 metal binding tag. Washed with 0.05% Triton X-100 and0.1% SDS in TBS, the membranes were further incubated with alkalinephosphatase-conjugated goat anti-mouse IgG secondary antibody, washedagain, and developed in BCIP-NTB substrate solution. The results weredocumented by imaging using a high resolution scanner.

Expression and Purification of HcTnI-C27 Fusion Protein and Recovery ofFree Peptides

Sequence-confirmed Tx3-SUMO-HcTnI-C27 and Tx3-SUMO-HcTnI-C27-Hexpression plasmids were used for transformation of BL21(DE3)pLysS E.coli competent cells. The fusion proteins were expressed in cultures inLB media upon isopropyl β-D-1-thiogalactopyranoside (IPTG) induction atmid-log phase of growth for 3 hours. Bacterial cells were then harvestedby centrifugation, resuspended with lysis buffer containing 6 M urea, 1M KC1, 20 mM phosphate buffer, pH 7.4, supplemented with 5 mM PMSF, andlysed using a French press. Lysate was centrifuged and the supernatantcontaining the fusion protein was loaded on a Zn(II) affinity columnequilibrated with the same buffer. The column was washed with five bedvolumes of lysis buffer and the Tx3-fusion protein was eluted with astep gradient of imidazole. The column fractions were examined withSDS-PAGE and the fusion protein peak was pooled, dialyzed againstde-ionized water, and concentrated by lyophilization. The lyophilizedfusion protein was resuspended in a minimal volume of SUMO cleavagebuffer (250 mM NaCl, 250 mM sucrose, 2 mM DTT, 40 mM Tris-HCl, pH 7.5).A 1:200 molar ratio of SUMO protease-to-fusion protein was used tocleave HcTnI-C27 and HcTnI-C27-H peptides at 4° C. overnight. Thecleaved product was loaded onto a re-equilibrated Zn(II) affinity columnand the flow-through containing free HcTnI-C27 or HcTnI-C27-H peptidewas collected, analyzed with small pore SDS-PAGE, dialyzed againstde-ionized water, and then concentrated by lyophilization. The fusionproteins and isolated free peptides were used in the followingstructural and function studies.

Synthetic HcTnI-C27 and HcTnI-C27-H Peptides

Free HcTnI-C27 and HcTnI-C27-H peptides were chemically synthesized at apurity of >98% using a commercial service. After verification of theirmAb TnI-1 epitope configuration and tropomyosin binding as describedherein, the chemically synthesized peptides were used in skinned musclecontractility studies.

Enzyme-Linked Immunosorbent Assay (ELISA)

The ELISA procedure used in the present study for epitope structure andprotein binding studies was performed as described in B.J. Biesiadeckiet al., Journal of Biomedicine and Biotechnology, (2011) 1-8. Briefly,96-well microtiter plates were coated with SUMO-HcTnI-C27 fusionprotein, SUMO-HcTnI-C27-H fusion protein, purified bovine cardiac TnI,or R192H mutant mouse cardiac TnI (2 μg/mL in Buffer A containing 0.1 MKC1, 3 mM MgCl₂, 10 mM PIPES, pH 7.0, 100 microliters/well) at 4° C.overnight. The plates were washed with Buffer T (0.05% Tween-20 inBuffer A) for 10 minutes and tapped dry. The wells were blocked usingBuffer B (1% BSA in Buffer T) at room temperature for one hour. Afterwashing again with Buffer T, serial dilutions of primary mAb against thecoated protein were added in triplicates in Buffer D (dilution buffer,0.1% BSA in Buffer T) and the plate was incubated at room temperaturefor two hours. After Buffer T washes, horseradish peroxidase(HRP)-conjugated anti-mouse immunoglobulin secondary antibody was addedto incubate at room temperature for one hour. After final washes,H202-2,2′-azinobis-(3-ethylbenzthiazolinesulfonic acid) substratesolution was added for colorimetric development at room temperature andthe plate was read using an automated microplate reader at 420 nm at 5minute intervals for 30 minutes.

The ELISA experiments were done in triplicate wells. Optical densitydata from the time point just before the end of the linear course ofcolor development were used to plot the titration curves. Eachexperiment was repeated one or more times to confirm the results.

Competitive ELISA

Competitive tropomyosin binding experiments were performed using aderivative protocol of the microtiter plate ELISA as described in B. J.Biesiadecki et al., Journal of Biomedicine and Biotechnology, (2011)1-8. Purified bovine cardiac a-tropomyosin was coated on 96-well plateat 5 μg/mL in Buffer A at 4° C. overnight. Washed and blocked as above,bovine cardiac TnI was added, at a predetermined concentration thatproduces sub-maximal binding for the immobilized tropomyo sin, to serialdilutions of HcTnI-C27 or HcTnI-C27-H peptides for incubation at roomtemperature for 2 hours. The competition between the cardiac TnIC-terminal peptides and intact cardiac TnI for tropomyosin binding wasmeasured via mAb 4H6 that recognizes an epitope in intact TnI but not inthe C-terminal end peptide, see S. Akhter et al., FEBS Open Bio 5 (2015)64-75, and HRP-anti-mouse IgG secondary antibody using standard ELISAprocedure as described above.

To compare the folding and conformation of chemically synthesizedHcTnI-C27 and HcTnI-C27-H peptides with that of the biologicallysynthesized counterparts, competitive ELISA affinity titrations againstmAb TnI-1 were performed. 96-well microtiter plates were coated withpurified bovine cardiac TnI in Buffer A and incubated at 4° C.overnight. Monoclonal antibody (mAb) TnI-1 was added, at a predeterminedconcentration that produces sub-maximal binding for the immobilizedbovine cardiac TnI, to serial dilutions of biologically or chemicallysynthesized HcTnI-C27 and HcTnI-C27-H peptides. The effects of thepeptides on competing with intact cardiac TnI for mAb TnI-1 weremeasured via HRP-anti-mouse IgG secondary antibody using standard ELISAprocedure as described above.

Contractility Measurements Using Membrane Permeabilized MusclePreparations

Extensor digitorum longus (EDL) muscles were obtained from adult C57B/L6mice immediately after euthanasia to prepare chemically permeabilizedmuscle preparations as described in J. Ochala et al., The FASEB Journal25(6) (2011) 1903-1913; and S. M. Roche et al., Journal of VisualizedExperiments 100 (2015). Briefly, whole EDL muscles were excised frommice and longitudinally dissected along fibers in a calcium freerelaxing buffer. The muscle strips dissected were washed with therelaxing buffer and stored in a 35 mm dish at −20° C. in 50% glycerolrelaxing-skinning solution to remove cellular membranes and theendogenous calcium handling system for use in the measurements ofcalcium sensitivity and force-pCa relationship.

Permeabilized rat and mouse left ventricular papillary musclepreparations were prepared using a skinned cryosection method. Papillarymuscles were dissected with a portion of ventricular free wall in oneend and valve tendon in the other end. The isolated papillary muscle waspinned down on a cork at the two ends using 30 gauge needles. A smalldrop of optimal cutting temperature (O.C.T.) compound was used to fillthe space between the muscle tissue and the surface of the cork beforeflash freezing by quickly submerging in liquid nitrogen. The frozenpapillary muscle was sectioned longitudinally at a thickness of 35 μmusing a cryostat and collected on a glass slide. Four stacked razorblades were used to cut the muscle sections longitudinally into 140-150μm wide strips. The muscle strips were washed in a relaxing buffer (BES40 mM, EGTA 10 mM, MgCl₂ 6.86 mM, ATP 5.96 mM, DTT 1 mM, creatinephosphate 33 mM, creatine kinase 200 U/mL, K-propionate 3.28 mM, pH 7.0,plus protease inhibitor cocktail) and then gently transferred intomyofibril relaxation buffer containing 50% glycerol in a 35 mm culturedish and stored at −20° C. for later use in force-pCa studies.

For contractility studies, the storage dish was placed on athermal-controlled metal stage at 0° C. under a dissection scope.Cryosectioned cardiac muscle strips selected with cardiomyocytes clearlyorganized along the long axis and EDL muscle fibers were mounted betweentwo aluminum T-clips and transferred to an 8-chamber thermo-controlledstage (802D, Aurora Scientific) on an inverted microscope in relaxationbuffer at 6-8° C. Seen through a 20× lens, the muscle preparation wasconnected to a force transducer (403A, Aurora Scientific) and a lengthcontroller (322-C, Aurora Scientific). The buffer was then switched to askinning solution (relaxation buffer containing 1% Triton X-100) for 20min to further permeabilize the muscle strips. After a wash withrelaxation buffer, the permeabilized muscle strip was placed in pCa 9.0buffer made by mixing the relaxing buffer (pCa 10.0) with an activationbuffer (BES 40 mM, EGTA 10 mM, MgCl₂ 6.64 mM, ATP 6.23 mM, DTT, 1 mM,CaCl₂ 10 mM, creatine phosphate 33 mM, creatine kinase 200 U/mL,K-propionate 2.09 mM, pH 7.0, plus protease inhibitor cocktail, pCa 4.0)and the sarcomere length was measured through a digital camera attachedto the microscope and adjusted to resting sarcomere length of 2.7 μm forEDL muscle fibers or 2.0 μm and 2.3 μm for cardiac muscle fibers.Calcium activated force was examined at pCa 6.5, 6.3, 6.0, 5.8, 5.5,5.0, and 4.5 at 15° C. using calcium-EGTA buffer sets made using aformula described in S. M. Roche et al., Journal of VisualizedExperiments 100 (2015). HcTnI-C27 peptide was then added at 20 μM andthe force-pCa measurements were repeated. The force-pCa curves wereplotted and fitted using Hill exponential equation for data analysis,see S.M. Roche et al., Journal of Visualized Experiments 100 (2015).

Data Analysis

Statistical analysis was performed using Student's t-test to comparepaired data points.

Results

Biological Synthesis of C-terminal Peptide of Human Cardiac TnI

The Tx3-SUMO-HcTnI-C27 fusion protein was readily expressed in E. coliand purified using a Zn(II) affinity column, FIG. 3A. Similarly highlevel expression and effective one-step purification was obtained forthe Tx3-SUMO-HcTnI-C27-H fusion protein, FIG. 3B.

While the purified Tx3-SUMO-HcTnI-C27 and Tx3-SUMO-HcTnIC27-H fusionproteins are both recognized by the anti-Tx3 tag mAb3C11 in Westernblot, only Tx3-SUMO-HcTnI-C27 was reactive to mAbTnI-1, FIG. 3B. Theresult is consistent with previous Western blot studies in intactcardiac TnI where the myopathic single amino acid R192H substitutioncompletely abolished the epitope recognized by mAb TnI-1, see Y. Li etal., Journal of Molecular and Cellular Cardiology 62 (2013) 227-236.

The results that the HcTnI-C27 peptide retains the mAb TnI-1 epitopestructure when fused with an unrelated carrier protein indicate itsretention of a native conformation. This epitope structure is preservedor intrinsically resumed after the denaturing process of SDS-PAGE andWestern blotting, FIG. 3B.

SUMO protease cleavage of the fusion proteins released the HcTnIC27 andHcTnI-C27-H peptides, FIG. 4A. The released peptides were separated fromthe carrier protein, any un-cleaved fusion protein and the protease,which all have the metal binding tag and were absorbed by thepost-cleavage Zn(II) column. The identities of the cleavage productswere verified by Western blotting using mAb 3C11 against the Tx3 tag andmAb TnI-1 against the HcTnI-C27 epitope, FIG. 4A. After cleavage fromthe fusion protein, the isolated HcTnI-C27 peptide remains reactive tomAb TnI-1 in Western blot, FIG. 4A, further demonstrating that thisshort peptide structure is able to configure the native epitopeconformation independently and after the denaturing process of SDS-PAGEand Western blotting, whereas the HcTnI-C27-H mutant peptide cleavedfrom the fusion protein remains non-reactive to mAb TnI-1, FIG. 4A.

The results demonstrate that the C-terminal end segment of TnI is astructural domain that forms the native conformation when isolated fromthe TnI backbone. This observation was confirmed by mAb TnI-1 Westernblot using chemically synthesized HcTnI-C27 peptide and HcTnI-C27-Hmutant peptide, FIG. 4B.

HcTnI-C27 Peptide Retains Native Configuration in Non-DenaturingConditions

To confirm the initial observation in Western blotting studies, ELISAtitrations further showed that Tx3-SUMO-HcTnI-C27 fusion protein reactswith mAb TnI-1 under non-denaturing conditions. The titration curve inFIG. 5A shows a saturable binding of mAb TnI-1 to Tx3-SUMO-HcTnI-C27fusion protein which is nearly identical to that of the coating controlusing anti-Tx3 tag mAb 3C11, FIG. 5B.

While Tx3-SUMO-HcTnI-C27-H lost binding to mAb TnI-1 under thepost-denaturing Western blotting condition, FIG. 4B, it showed a clearlydetectable but significantly decreased binding to mAb TnI-1 in the ELISAtitration using non-denaturing conditions, FIG. 5A. This resultdemonstrates that while the myopathic R192H mutation alters theconformation and function of the C-terminal end segment of cardiac TnI,the mAb TnI-1 epitope is partially preserved in the Tx3-SUMO-HcTnI-C27-Hfusion protein.

The retained overall native conformation of HcTnI-C27 peptide in thefusion protein and the alteration due to the R192H mutation were furthercompared to that of the C-terminal end segment residing in situ inintact cardiac TnI. The results of mAb TnI-1 titration in FIG. 6 showedthat the binding affinity of Tx3-SUMO-HcTnI-C27 fusion protein for mAbTnI-1 is similar to that of intact wild type bovine cardiac TnI whereasthe R192H mutation produced similar decreases in the affinity for mAbTnI-1 in Tx3-SUMO-HcTnI-C27-H fusion protein and in intact mouse cardiacTnI engineered with the RCM mutation. The similar epitope conformationof HcTnI-C27 peptide in fusion with an unrelated carrier protein and insitu in cardiac TnI further demonstrates its nature as an independentstructural domain in troponin complex.

HcTnI-C27 Peptide Retains the Property of Binding Tropomyosin

When present in a troponin complex, the C-terminal end segment of TnIpossesses a Ca²±-regulated, relatively low-affinity but saturablebinding to tropomyosin. Demonstrating that this physiologic activity isretained with the isolated HcTnI-C27 peptide, the results of competitivetropomyosin binding test in FIG. 7 showed that the presence of HcTnI-C27peptide in solution resulted in a dose-dependent competition with intactbovine cardiac TnI for the binding to tropomyosin immobilized onmicrotiter plate. This function was diminished with the HcTnI-C27-Hmutant peptide. The results demonstrate a preserved physiologicalfunction of HcTnI-C27 peptide in isolation from the TnI backbone.

Chemically Synthesized HcTnI-C27 and HcTnI-C27-H Peptides ExhibitEpitope Conformations Similar to their Biological-Made Counterparts inPhysiologic Solution

Upon demonstrating the preserved native conformation and biochemicalactivity of HcTnI-C27 and HcTnI-C27-H peptides made from bacterialexpression, chemically synthesized peptides were evaluated in order touse them in further structural and functional studies. The competitiveELISA titration results in FIG. 8A demonstrated that synthetic andbiological-made HcTnI-C27 peptides had nearly identical effectiveness oncompeting in physiologic solution with intact cardiac TnI coated onmicrotiter plate for the binding of mAb TnI-1. Consistently, chemicallysynthesized and biologically made HcTnI-C27-H mutant peptides bothdrastically lost the binding affinity for mAb TnI-1, FIG. 8B.

HcTnI-C27 Peptide Reduces Ca²⁺ Sensitivity of Skinned Muscle Strips

Since the C-terminal end segment of TnI is conserved in cardiac andskeletal muscle isoforms of TnI, see FIG. 1, HcTnI-C27 peptide wastested for its effect on the contractility of skinned mouse EDL musclefibers. The results in FIG. 9 showed that at physiologic sarcomerelength of 2.7 μm, the presence of 20 μM HcTnI-C27 peptide produced amyofilament Ca²⁺-desensitization effect predominantly in the activatedstate with significantly decreased cooperativity. The maximum forceproduction was not affected by the addition of HcTnI-C27 peptide.Reproduction of the physiological effect of HcTnI-C27 peptide in askeletal muscle preparation adds evidence for the conserved function ofthe C-terminal end segment of TnI.

To investigate the function of the C-terminal end segment of TnI inregulating muscle contraction, the effect of wild type HcTnI-C27 peptideon the force-pCa relationship of cardiac muscle was first studied usingskinned rat ventricular papillary muscle sections at sarcomere lengthsof 2.0 μm and 2.3 μm. The contractility results in FIG. 10A showed thatHcTnI-C27 peptide had a small effect on lowering pCa50 and cooperativityat sarcomere lengths of 2.0 μm. However, it produced a statisticallysignificant right-shift of pCa50 at sarcomere lengths of 2.3 μm with atrend of decreasing cooperativity, FIG. 10B. The maximum forceproduction was not affected by the addition of HcTnIC27 peptide, see thebar graphs in FIGS. 10A and 10B. It is interesting that the effect ofHcTnI-C27 on Ca²⁺-desensitization and the effect of increasing sarcomerelength on increasing Ca²⁺-sensitivity are both more notable at higher[Ca²⁺ in the isometric contractility assay, corresponding to theactivated state of myofilaments, FIG. 10B. As an outcome, the effect ofHcTnI-C27 peptide diminished the Ca²⁺-sensitization effect of increasingsarcomere length from 2.0 μm to 2.3 μm, FIG. 10B. The resultsdemonstrate that the addition of free HcTnI-C27 peptide to skinnedmuscle preparation produces a myofilament activation—as well assarcomere length-dependent Ca²⁺-desensitization.

The effect of HcTnI-C27 peptide on myofilament Ca²⁺-sensitivity andforce development was confirmed using skinned mouse ventricularpapillary muscle preparations. The results in FIGS. 11A and 11B showedthat the addition of HcTnI-C27 peptide reproduced theCa²⁺-desensitization effect with right-shifts of the force-pCa curves inskinned mouse cardiac muscle strips. Different from that seen in ratcardiac muscle (FIGS. 10A and 10B), the effect was more obvious atsarcomere length of 2.0 μm in the activated state with significantlylowered cooperativity in comparison to that at sarcomere length of 2.3μm (FIGS. 11A and 11B). One possible explanation for the differentsarcomere length dependence of HcTnI-C27's Ca²⁺ desensitization effecton mouse and rat cardiac muscles could be a species-specific differencein cardiomyocyte compliance, which is known to affect myofilamentforce-pCa relationships. Supporting this notion, we detected a trend ofmore increase in the passive tension of skinned mouse cardiac muscle atpCa 9 when increasing the sarcomere length from 2.0 μm to 2.3 μm thanthat in the rat cardiac muscle preparations. The higher passive tensionreflects a lower compliance of mouse cardiac muscle than that of rat,which may confer different myofilament responses to the changes ofsarcomere length.

When added to skinned cardiac (FIGS. 9 and 10) and skeletal (FIG. 11)muscle preparations, free HcTnI-C27 peptide produces aCa²⁺-desensitization effect without significant effect on maximum forceproduction.

The effect of additional free HcTnI-C27 peptide may reflect anartificial augmentation of the in situ inhibitory function of theC-terminal end domain of TnI in facilitating myocardial relaxation. Itseffect on decreasing the cooperativity of force-pCa curves reflects aselective decrease of myofilament Ca²⁺-sensitivity at the higher Ca²⁺concentrations corresponding to the activated state. This new functionalfeature of the C-terminal end segment of TnI indicates a novelmyofilament mechanism to modulate the kinetics of muscle contractility.The post-activation Ca²⁺-desensitization of myofibrils lays a basis forslowing down late-systolic velocity to prolong ventricular ejection timewithout reducing maximum force development.

Abbreviations

-   cTnI: cardiac troponin subunit I-   HcTnI-C27: the C-terminal 27 amino acid peptide of human cardiac TnI-   HcTnI-C27-H: R192H mutant of the C-terminal 27 amino acid peptide of    human cardiac TnI-   pCa50: Ca²⁺ concentration for 50% maximum force-   SUMO : small ubiquitin-like modifier-   Tx3: a transition metal-binding tag-   EDL: extensor digitorum longus

cDNA sequences:cDNA sequence encoding SEQ ID NO: 1 (derived from NM_000363.5)(SEQ ID NO: 12)gaggtgggagactggcgcaagaacatcgatgcactgagtggaatggagggccgcaagaaaaagtttgagcDNA sequence encoding SEQ ID NO: 3 (derived from NM_000363.5)(SEQ ID NO: 13)gaaaaccgggaggtgggagactggcgcaagaacatcgatgcactgagtggaatggagggccgcaagaaaaagtttgagagc cDNA sequence encoding SEQ ID NO: 4 (derived from NM_001146587.1)(SEQ ID NO: 14)aagaaagaagaggtcactgactggcgtcaaaatgtggatgccatgtctggcatggagggcagaaagaagatgtttgacgctcDNA sequence encoding SEQ ID NO: 5 (derived from AY773673.1)(SEQ ID NO: 15)gaaatacgtgaagttggagactggagaaagaacgtcgatgctctcagcggcatggagggcaggaaaaagaaatttgaatca cDNA sequence encoding SEQ ID NO: 6 (derived from AY773673.1)(SEQ ID NO: 16)gaaatacgtgaagttggagactggagaaagaacgtcgatgctctcagcggcatggagggcaggaaaaagaaatttgaatcatctggagcagtgcaaact cDNA sequence encoding SEQ ID NO: 7 (derived from NM_213570.1)(SEQ ID NO: 17)gagagccgcgaggtcggcgattggcggaagaacgtggacgcgctgagcggcatggaggggcgcaagaagaagttcgaggcgcDNA sequence encoding SEQ ID NO: 8 (derived from NM_213570.1)(SEQ ID NO: 18)gagagccgcgaggtcggcgattggcggaagaacgtggacgcgctgagcggcatggaggggcgcaagaagaagttcgaggcgccggggggggggcagggcMouse cDNA sequence encoding SEQ ID NO: 9 (derived from NM_009406)(SEQ ID NO: 19)Gaaaaccgggaggtgggagactggcgcaagaatatcgatgcactgagtggcatggaaggccgcaagaaaaagtttgagggc Bovine cDNA sequence encoding SEQ ID NO: 9 (derived from NM_001040517)(SEQ ID NO: 20)Gaaaaccgagaggtgggagactggcgcaagaacattgacgcgttgagtggaatggaaggccgcaagaagaagtttgagggc cDNA sequence encoding SEQ ID NO: 10 (derived from CR541996)(SEQ ID NO: 21)cggcctgtggaggtgggtgactggaggaagaacgtggaggccatgtctggcatggaaggccggaagaagatgtttgatgccgccaatgctccgacctcacaa cDNA sequence encoding SEQ ID NO: 11 (derived from NM_001145829)(SEQ ID NO: 22)gacctgcgagacgtgggtgactggaggaagaacatcgaggagaagtctggcatggagggccggaagaagatgtttgagtccgagtcc

Any patents or publications mentioned in this specification areincorporated herein by reference to the same extent as if eachindividual publication is specifically and individually indicated to beincorporated by reference.

The compositions and methods described herein are presentlyrepresentative of preferred embodiments, exemplary, and not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art. Such changes and other usescan be made without departing from the scope of the invention as setforth in the claims.

1. A method of treating a disorder of cardiac muscle and/or skeletalmuscle in a subject, comprising: administering a therapeuticallyeffective dose of a C-terminal portion of troponin I capable ofreduction of cardiac muscle and/or skeletal muscle sensitivity to Ca²⁺without decreasing maximum force production.
 2. The method of claim 1,wherein the C-terminal portion of troponin I comprises a peptideselected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:32, or a variant ofany thereof.
 3. The method of claim 2, wherein the variant comprises oneor more conservative amino acid substitutions and is capable ofreduction of cardiac muscle and/or skeletal muscle sensitivity to Ca²⁺without decreasing maximum force production.
 4. The method of claim 2,wherein the variant comprises one or more conservative amino acidsubstitutions, wherein the variant has at least 70% identity, at least80% identity, at least 90% identity, or at least 95% identity to any of:SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,and SEQ ID NO:32, and wherein the variant is capable of reduction ofcardiac muscle and/or skeletal muscle sensitivity to Ca²⁺ withoutdecreasing maximum force production.
 5. The method of claim 2, whereinthe variant has at least 22 amino acids, wherein the variant comprisesone or more conservative amino acid substitutions compared to SEQ IDNO:32, wherein the variant has at least 70% identity, at least 80%identity, at least 90% identity, or at least 95% identity to SEQ IDNO:32, and wherein the variant is capable of reduction of cardiac muscleand/or skeletal muscle sensitivity to Ca²+without decreasing maximumforce production.
 6. The method of claim 2, wherein the variant has atleast 23 amino acids, wherein the variant comprises one or moreconservative amino acid substitutions compared to SEQ ID NO:1, whereinthe variant has at least 70% identity, at least 80% identity, at least90% identity, or at least 95% identity to SEQ ID NO:1, and wherein thevariant is capable of reduction of cardiac muscle and/or skeletal musclesensitivity to Ca²⁺ without decreasing maximum force production.
 7. Themethod of claim 2, wherein the variant has at least 27 amino acids,wherein the variant comprises one or more conservative amino acidsubstitutions compared to SEQ ID NO:3, wherein the variant has at least70% identity, at least 80% identity, at least 90% identity, or at least95% identity to SEQ ID NO:3, and wherein the variant is capable ofreduction of cardiac muscle and/or skeletal muscle sensitivity to Ca²⁺without decreasing maximum force production.
 8. The method of claim 1,wherein the subject is human.
 9. The method of claim 1, furthercomprising administering an additional therapeutic agent.
 10. The methodof claim 1, wherein administering a therapeutically effective dose of aC-terminal portion of troponin I, comprises administering an expressioncassette comprising a nucleic acid encoding the C-terminal portion oftroponin I, operably linked to a promoter.
 11. The method of claim 10,wherein the promoter is capable of driving expression of the nucleicacid encoding the C-terminal portion of troponin I in cardiac muscleand/or skeletal muscle,
 12. The method of claim 10, wherein the promoteris a cardiac muscle protein promoter or a skeletal muscle proteinpromoter.
 13. The method of claim 1, wherein the subject has heartfailure.
 14. The method of claim 1, wherein the subject has a cardiacdisorder relating to mutation of troponin or other sarcomeric proteins,such as hypertrophic cardiomyopathy (HCM), restrictive cardiomyopathy(RCM), and dilated cardiomyopathy (DCM), and/or a coordination disorderof the skeletal muscles.
 15. A pharmaceutical composition, comprising: aC-terminal portion of troponin I of claim 1 capable of reduction ofcardiac muscle and/or skeletal muscle sensitivity to Ca²⁺ withoutdecreasing maximum force production.
 16. A pharmaceutical composition,comprising: a nucleic acid encoding the C-terminal portion of troponin Iof claim 1 capable of reduction of cardiac muscle and/or skeletal musclesensitivity to Ca²⁺ without decreasing maximum force production.
 17. Thepharmaceutical composition of claim 16, wherein the nucleic acid isincluded in an expression cassette and is operably linked to a promoter.18. The pharmaceutical composition of claim 17, wherein the promoter iscapable of driving expression of the nucleic acid encoding theC-terminal portion of troponin I in cardiac muscle and/or skeletalmuscle,
 19. The pharmaceutical composition of claim 17, wherein theexpression cassette is included in a vector.
 20. An assay foridentification of a test compound capable of capable of reduction ofcardiac muscle and/or skeletal muscle sensitivity to Ca²⁺ withoutdecreasing maximum force production comprising: contacting humanalpha-tropomyosin, or a non-human homologue thereof, with a testcompound under conditions that promote specific interaction between thetropomyosin and the test compound; and detecting a change in cardiacmuscle and/or skeletal muscle sensitivity to Ca²⁺ without decreasingmaximum force production, if present.